Anti-inflammatory compounds and uses thereof

ABSTRACT

The present invention provides anti-inflammatory compounds, pharmaceutical compositions thereof, and methods of use thereof for treating inflammatory disorders. The present invention also provides methods of identifying anti-inflammatory compounds and methods of inhibiting NF-κB-dependent target gene expression in a cell.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/439,528, filed on May 22, 2006, and issued as U.S. Pat. No.7,534,858, which is a continuation of U.S. patent application Ser. No.09/847,946, filed on May 2, 2001 and issued as U.S. Pat. No. 7,049,395,which, in turns, claims priority to U.S. Provisional Patent ApplicationSer. No. 60/201,261 filed May 2, 2000 and to U.S. patent applicationSer. No. 09/643,260, filed Aug. 22, 2000 and issued as U.S. Pat. No.6,864,355. The entire contents of each of the foregoing are expresslyincorporated herein by reference.

U.S. GOVERNMENT SUPPORT

This work was supported by a grant from the National Institute of Health(AI33443).

FIELD OF THE INVENTION

The invention relates to compositions and methods for the selectiveinhibition of cytokine-mediated NF-κB activation by blocking theinteraction of NEMO with IκB kinase-β (IKKβ) at the NEMO binding domain(NBD). The blockade of IKKβ-NEMO interaction results in inhibition ofIKKβ kinase activation and subsequent decreased phosphorylation of IκB.Phosphorylation of IκB is an integral step in cytokine-mediated NF-κBactivation.

BACKGROUND OF THE INVENTION

NF-κB is a transcription factor which mediates extracellular signalsresponsible for induction of genes involved in pro-inflammatoryresponses (Baltimore et al., (1998) U.S. Pat. No. 5,804,374). NF-κB isanchored in the cytoplasm of most non-stimulated cells by a non-covalentinteraction with one of several inhibitory proteins known as IκBs (May &Ghosh, (1997) Semin. Cancer. Biol. 8, 63-73; May & Ghosh, (1998)Immunol. Today 19, 80-88; Ghosh et al., (1998) Annu. Rev. Immunol. 16,225-260). Cellular stimuli associated with pro-inflammatory responsessuch as TNFα, activate kinases, which in turn activate NF-κB byphosphorylating IκBs. The kinases that phosphorylate IκBs are called IκBkinases (IKKs).

Phosphorylation targets IκBs for ubiquitination and degradation. Thedegradation and subsequent dissociation of IκBs from NF-κB reveals thenuclear localization signal on NF-κB, resulting in nuclear translocationof active NF-κB, leading to up-regulation of genes responsive to NF-κB(May & Ghosh, (1997) Semin. Cancer. Biol. 8, 63-73; May & Ghosh, (1998)Immunol. Today 19, 80-88; Ghosh et al., (1998) Annu. Rev. Immunol. 16,225-260; Siebenlist et al., (1994) Annu. Rev. Cell Biol. 12, 405-455).Phosphorylation of IκBs is therefore an essential step in the regulationof NF-κB mediated pro-inflammatory responses.

The identification and characterization of kinases that phosphorylateIκBs has led to a better understanding of signaling pathways involvingNF-κB activation. Several different subtypes of IKK have been identifiedthus far. IKKα was initially identified as an IκB kinase induced by TNFαstimulation in HeLa cells (DiDonato et al., (1997) Nature 388, 548-554).Another IκB kinase homologous to IKKα was identified, termed IKKβ anddetermined to be the major IκB kinase induced following TNFα stimulation(Takeda et al., (1999) Science 284, 313-316; Hu et al., (1999) Science284, 316-320; Li et al., (1999) Science 284, 321-325; Pot et al., (2000)U.S. Pat. No. 6,030,834; Woronicz & Goeddel (1999) U.S. Pat. No.5,939,302). IKKα and IKKβ have an overall homology of 52% and a 65%homology in the kinase domain (Zandi et al., (1997) Cell 91, 243-252).

IκB protein kinases (IKKs) phosphorylate IκBs at specific serineresidues. For example, they specifically phosphorylate serines 32 and 36of IκBα (Traenckner et al., (1995) EMBO J. 14, 2876-2883; DiDonato etal., (1996) Mol. Cell. Biol. 16, 1295-1304). Phosphorylation of bothsites is required to efficiently target IκBα for degradation.Furthermore, activation of IKKα and IKKβ is usually in response to NF-κBactivating agents and mutant IKKα and IKKβ, which are catalyticallyinactive, can be used to block NF-κB stimulation by cytokines such asTNFα and IL-1 (Régnier et al., (1997) Cell 90, 373-383; Delhase et al.,(1999) Science 284, 309-313). IκB protein kinases are thereforeessential in the regulation of NF-κB activation processes.

IKKα and IKKβ have distinct structural motifs including an aminoterminal serine-threonine kinase domain separated from a carboxylproximal helix-loop-helix (H-L-H) domain by a leucine zipper domain.These structural characteristics are unlike other kinases, and thenon-catalytic domains are thought to be involved in protein-proteininteractions. Proteins which bind to IKKs may therefore be capable ofregulating the activity of NF-κB (Marcu et al., (1999) U.S. Pat. No.5,972,655) and potentially regulating downstream events such asinduction of NF-κB. For instance, NEMO (NF-κB Essential Modulator) is aprotein which has been identified to bind to IKKs and facilitate kinaseactivity (Yamaoke et al., (1998) Cell 93, 1231-1240; Rothwarf et al.,(1998) Nature 395, 287-300; Mercurio et al., (1999) Mol. Cell. Biol. 19,1526-1538; Haraj & Sun, (1999) J. Biol. Chem. 274, 22911-22914; Jin &Jeang, (1999) J. Biomed. Sci. 6, 115-120).

Inflammation is defined as the reaction of vascularized living tissue toinjury. As such, inflammation is a fundamental, stereotyped complex ofcytologic and chemical reactions of affected blood vessels and adjacenttissues in response to an injury or abnormal stimulation caused by aphysical, chemical or biological agent. Inflammation usually leads tothe accumulation of fluid and blood cells at the site of injury, and isusually a healing process. However, inflammation sometimes causes harm,usually through a dysfunction of the normal progress of inflammation.Inflammatory diseases are those pertaining to, characterized by,causing, resulting from, or becoming affected by inflammation. Examplesof inflammatory diseases or disorders include, without limitation,asthma, lung inflammation, chronic granulomatous diseases such astuberculosis, leprosy, sarcoidosis, and silicosis, nephritis,amyloidosis, rheumatoid arthritis, ankylosing spondylitis, chronicbronchitis, scleroderma, lupus, polymyositis, appendicitis, inflammatorybowel disease, ulcers, Sjorgen's syndrome, Reiter's syndrome, psoriasis,pelvic inflammatory disease, orbital inflammatory disease, thromboticdisease, and inappropriate allergic responses to environmental stimulisuch as poison ivy, pollen, insect stings and certain foods, includingatopic dermatitis and contact dermatitis.

Inflammatory diseases present a worldwide problem. Studies of diseaseburden have re-affirmed that tuberculosis is among the top 10 causes ofdeath in the world. Asthma affects 5% of the adult population and 10-15%of the population of children (Armetti and Nicosia (1999) Boll Chim.Farm. 138(11):599). Asthma is a chronic inflammatory disease that isassociated with widespread but variable airflow obstruction.

Sepsis is yet another inflammation disorder and is caused by thepresence of various pus-forming and other pathogenic microbes, or theirtoxins, in the blood or tissues of a subject. Sepsis is characterized bya systemic inflammatory response to bacterial products during infection.The symptoms of sepsis, such as fever, are caused at least in part bythe inflammatory response of the body to the infecting agent.

Accordingly, there is still a great need for compounds useful fortreating inflammatory disorders.

SUMMARY OF THE INVENTION

The present invention provides anti-inflammatory compounds,pharmaceutical compositions thereof, and methods of use thereof fortreating inflammatory disorders. The present invention is based, atleast in part, on the identification of the NEMO binding domain (NBD) onIκB kinase-α (IKKα) and on IκB kinase-β (IKKβ).

Accordingly, in one aspect, the present invention providesanti-inflammatory compounds comprising a NEMO binding domain (NBD).

In one embodiment, the present invention provides anti-inflammatorycompounds comprising fusions of a NEMO binding domain and at least onemembrane translocation domain. In a preferred embodiment, the membranetranslocation domain facilitates membrane translocation of theanti-inflammatory compounds of the invention in vivo. The membranetranslocation domain may, for example, be the third helix of theantennapedia homeodomain or the HIV-1 Tat protein. In one embodiment,the NEMO binding domain is a polypeptide having the sequence set forthin SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,or 19.

In another embodiment, the present invention provides anti-inflammatorycompounds comprising: (a) peptides which include, or consist of, theamino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18 or 19; (b) a fragment of at least three amino acidsof the amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18 or 19; (c) peptides which include aconservative amino acid substitution of the amino acid sequences of SEQID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19;and (d) naturally occurring amino acid sequence variants of the aminoacid sequences of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or 19.

In another aspect, this invention provides pharmaceutical compositionscomprising the anti-inflammatory compounds of the invention, e.g.,pharmaceutical compositions which include one or more pharmaceuticallyacceptable carriers.

In yet another aspect, the invention features a method of treating aninflammatory disorder, e.g., asthma, lung inflammation or cancer, in asubject. The method includes administering to the subject atherapeutically effective amount of one or more anti-inflammatorycompounds of the invention. Without intending to be limited bymechanism, it is believed that the anti-inflammatory compounds of theinvention may act (directly or indirectly) by blocking the recruitmentof leukocytes into sites of acute and chronic inflammation, bydown-regulating the expression of E-selectin on leukocytes, or byblocking osteoclast differentiation.

In another aspect, the present invention provides a method of inhibitingNF-κB-dependent target gene, e.g., E-selectin, expression in a cell. Themethod includes contacting a cell with an anti-inflammatory compound ofthe present invention, thereby inhibiting NF-κB-dependent target geneexpression in a cell.

In yet another aspect, the present invention provides methods ofinhibiting NF-κB induction (e.g., IKKα and/or IKKβ dependent induction)in a cell by contacting a cell with an effective amount of ananti-inflammatory compound of the present invention, thereby inhibitingNF-κB induction in a cell. In one embodiment of this invention, suchmethods utilize anti-inflammatory compounds which include at least onemembrane translocation domain. In still another specific embodiment ofthis invention, the anti-inflammatory compound s utilized in suchmethods include amino acid sequences comprising the sequences of SEQ IDNO:2, 4, 5, 6, 11, 12, 16, 17 or 18.

In another aspect, the present invention provides methods of identifyingan anti-inflammatory compound. The methods include exposing cells whichexpress NEMO and NF-κB to a test compound; and determining whether thetest compound modulates activation of NF-κB by the cell, therebyidentifying an anti-inflammatory compound.

In another aspect, the present invention provides methods of identifyingan anti-inflammatory compound by exposing cells which express NEMO to atest compound; and determining whether the test compound modulates anactivity of NEMO, thereby identifying an anti-inflammatory compound,e.g., a compound which modulates the activity of NEMO.

One particular advantage of the anti-inflammatory compounds of thepresent invention is that while blocking NF-κB induction via IKK, theydo not inhibit the basal activity of NF-κB.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts results from experiments indicating that NEMO interactswith the COOH-terminus of IKKβ. (A) GST alone or GST-NEMO wereprecipitated from bacterial lysates using glutathione-agarose, separatedby SDS-PAGE (10%) and the gel was stained with Coomassie blue (leftpanel). Equal amounts of GST or GST-NEMO were used in subsequent GSTpull-down experiments. The scheme depicted in the right panel representsthe COOH- and NH2-terminal truncation mutants of IKKβ used to determinethe region of NEMO interaction. (B) IKKβ mutants were cloned, expressedby in vitro translation (input; left panel) and used for GST pull-down(right panel). (C) Wild-type IKKβ and IKKβ-(644-756) were in vitrotranslated (left panel) and used for GST pull-down analysis (leftpanel). (D) HeLa cells were transiently transfected with either vectoralone or increasing concentrations (0.25, 0.5, 1.0 μg/ml) of thexpress-tagged IKKβ-(644-756) construct together with thepBIIX-luciferase reporter plasmid. After forty-eight hours cells weretreated with either TNFα (10 ng/ml) or IL-1β (10 ng/ml) for four hoursthen NF-κB activity was measured. Western blot analysis from portions ofthe lysate using anti-xpress (inset) demonstrates the increasing levelsof expressed protein.

FIG. 2 depicts results from experiments indicating that the firstα-helical region of NEMO is required for binding to IKKβ. (A) Atruncated version of IKKβ consisting of only the COOH-terminus fromresidue V644 to S756 was fused with GST (GST-644-756) and expressed inbacteria. After precipitation by glutathione agarose, GST alone andGST-(644-756) were separated by SDS-PAGE (10%) and the gel was stainedwith Coomassie blue (left panel). Equal amounts of each protein wereused for subsequent GST pull-down analyses. Various NH2- andCOOH-terminal truncations of NEMO were constructed, [³⁵S]-methioninelabeled and used for in vitro pull down (right panel). Mutants thatinteracted with GST-(644-756) are indicated (+). None of the mutantsinteracted with GST alone. (B) Wild-type NEMO and a deletion mutantlacking the first α-helical region (del.αH) were in vitro translated(left panel: input) and used for GST pull-down using the proteins shownabove (A: left). (C) HeLa cells were transfected with pBIIx-luciferasetogether with either pcDNA-3 (vector) or increasing concentrations ofdel.αH (0.25, 0.5, 1.0 μg/ml) for forty-eight hours then treated forfour hours with TNFα (10 ng/ml). Cells were then lysed and NF-κBactivity was measured by luciferase assay.

FIG. 3 depicts results from experiments indicating that interaction withNEMO and functional kinase activity requires an IKKα-homologous regionof the IKKβ COOH-terminus. (A) Truncation mutations of IKKβ sequentiallyomitting the extreme COOH-terminus (1-733), the serine-free region(1-707), the serine rich-domain (1-662) and the α1-region (1-644) wereexpressed and labeled by in vitro translation and used for GST pull-downby GST-NEMO (FIG. 1A). None of the mutants interacted with GST alone.(B) Sequence alignment of the extreme COOH-termini of IKKβ and IKKα. Theα2- and glutamate-rich regions are indicated and the six identical aminoacids are shaded. (C) Wild-type IKKβ and the truncation mutants (1-733and 1-744) were [³⁵S]-methionine-labeled (input) and used for in vitropull down with either GST alone or GST-NEMO. (D) HeLa cells weretransfected for forty-eight hours with 1 μg/well of the indicatedFLAG-tagged constructs followed by immunoprecipitation using anti-FLAG.The immunoprecipitates were incubated in kinase buffer containing[³²P]-labeled γATP for fifteen minutes at 30° C. then washed with lysisbuffer containing 1% Triton-100. Resulting complexes were separated bySDS-PAGE (10%) and visualized by autoradiography (upper panel). Thelower panel is an immunoblot from identical samples demonstratingequivalent amounts of transfected protein in each lane. (E) HeLa cellswere transfected for 48 hours with 1 μg/ml of the indicated constructsor empty vector (pcDNA-3) together with pBIIx-luciferase. NF-κB activitywas determined by luciferase assay. (F) HeLa cells transfected forforty-eight hours with FLAG-tagged versions of either IKKβ (wild-type)or IKKβ-(1-733) were either untreated (−) or treated for seven minutes(+) with TNFα. (10 ng/ml). Following lysis and immunoprecipitation usinganti-FLAG, immune-complex kinase assay (upper panels) was performed.Identical samples were immunoprecipitated and immunoblotted withanti-FLAG (lower panels).

FIG. 4 depicts results from experiments indicating that association ofNEMO with IKKβ and IKKα reveals the NEMO binding domain (NBD) to be sixCOOH-terminal amino acids. (A) COS cells transiently transfected withvector alone, FLAG-tagged IKKα or IKKβ (1 μg/well) or xpress-tagged NEMO(1 μg/well) to a total DNA concentration of 2 μg/well as indicated.Following lysis, immunoprecipitations (IP) were performed usinganti-FLAG (M2) and the contents of precipitates visualized byimmunoblotting (IB) with either anti-FLAG (M2) or anti-xpress. A portionof pre-IP lysate was immunoblotted with anti-xpress to ensure equivalentlevels of NEMO expression in transfected cells. (B) Wild-type IKKα andIKKα-(1-737) were expressed and labeled (input) and used for GSTpull-down using GST or GST-NEMO. (C) Full length cDNA encoding humanIKKi was obtained by RT-PCR from HeLa cell mRNA using the Expand™ LongTemplate PCR System (Boehringer Mannheim), the forward primer(5′-CTAGTCGAATTCACCATGCAGAGCACAGCCAATTAC) (SEQ ID NO: 22) and thereverse primer (3′-CTAGTCTCTAGATTAGACATCAGGAGGTGCTGG) (SEQ ID NO: 23)and cloned into the EcoRI and XbaI sites of pcDNA-3. GST pull-downanalysis was performed using [³⁵S]-methionine-labeled IKKα, IKKβ andIKKi. (D) A deletion mutant of IKKβ lacking the NBD (del.NBD) was[³⁵S]-methionine-labeled (input) and used for GST pull down analysis.(E) A Fauchere-Pliska hydrophobicity plot of the COOH-terminus(N721-S756) of human IKKβ was generated using MacVector (version 6.5.3)software. The NBD (L737-L742) is boxed. (F) COS cells were transfectedfor forty-eight hours with a total of 2 μg DNA/well of either vectoralone, vector plus NEMO-FLAG or NEMO-FLAG plus xpress-tagged versions ofIKKβ-(1-744) containing point mutations within the NBD as indicated.Following lysis and immunoprecipitation using anti-FLAG (M2), immunoblotanalysis was performed with either anti-FLAG or anti-xpress. The levelof expressed protein in pre-IP lysate was determined by immunoblottingwith anti-xpress (lower panel). (G) HeLa cells were transientlytransfected for forty-eight hours with the indicated constructs togetherwith pBIIX-luciferase and NFκB activity in lysate was measured byluciferase assay.

FIG. 5 depicts results from experiments indicating that a cell-permeablepeptide spanning the IKKβ NBD inhibits the IKKβ/NEMO interaction,TNFα-induced NF-κB activation and NF-κB-dependent gene expression. (A)GST-pull-down analysis was performed using either GST-NEMO-in vitrotranslated IKKβ (upper panel) or GST-IKKβ-(644-756)-in vitro translatedNEMO (lower panel). The assay was performed in the absence (no peptide)or presence of increasing concentrations (125, 250, 500 or 1000 μM) ofeither mutant (MUT) or wild-type (WT) NBD peptide. (B) HeLa cells wereincubated with either peptide (200 μM) for the times indicated.Following lysis, the IKK complex was immunoprecipitated using anti-NEMOand the resulting immunoblot probed with anti-IKKβ. (C) HeLa cells weretransfected for forty-eight hours with pBIIX-luciferase then incubatedfor two hours in the absence (control) or presence of mutant orwild-type NBD peptide (100 and 200 μM of each). Subsequently the cellswere either treated with TNFα (10 ng/ml) as indicated (left panel) orleft untreated (right panel) for a further four hours after which NF-κBactivation was measured by luciferase assay. (D) HeLa cells wereincubated for three hours with increasing concentrations (50, 100 or 200μM) of each peptide followed by treatment for fifteen minutes with TNFα(10 ng/ml) as indicated (+). Following lysis, nuclear extracts were madeand 10 μg of protein from each sample was used for EMSA using a specific[³²P]-labeled KB-site probe. (E) Primary HUVEC were pre-incubated fortwo hours with of wild-type (left) or mutant (right) NBD peptides (100μM) then stimulated with TNFα (10 ng/ml) for a further six hours.Control cells received no peptide. Cells were stained with eitheranti-E-selectin (H4/18) or a non-binding control antibody (K16/16) andexpression was measured by FACS (FACSort, Becton Dickinson). Theprofiles show E-selectin staining in the absence (shaded) and presence(solid line) of TNFα and control antibody staining under the sameconditions (dashed line, no TNFα; dotted line, TNFα).

FIG. 6 depicts results from experiments indicating that the wild-typeNBD peptide inhibits NF-κB-induced gene expression and experimentallyinduced inflammation. (A) PMA-induced ear edema in mice topicallytreated with either vehicle (VEH), dexamethasone (DEX) or NBD peptideswas induced and measured as described in Example 8. Data are presentedas mean differences in ear thickness±SD (*=p<0.05 compared with bothuntreated control [−] and vehicle [VEH]). (B) The effects of the NBDpeptide compared with the effect of dexamethasone (DEX) on Zymosan(ZYM)-induced peritonitis in mice were determined as described again inExample 8. Control mice were injected with phosphate-buffered saline(PBS).

FIG. 7 depicts results from experiments indicating the dose dependentinhibition of osteoclast differentiation by wild-type but not mutant NBDpeptides. Data are presented as the mean determination of triplicatesamples±SD.

FIG. 8 depicts the results of a mutational analysis of D738 within theNEMO binding domain (NBD) of human IKKβ. (A) The aspartic acid residueat position 738 of IKKβ was substituted with either alanine, asparagineor glutamic acid using PCR-mutagenesis. (B) The IKKβ (D738) mutantsshown in A were ³⁵S-methionine-labeled by in vitro transcription andtranslation then used for GST pull-down analysis using GST-NEMO aspreviously described. (C) Hela cells were transiently transfected usingthe Fugene6 transfection method with the NF-κB-dependent reporterconstruct pBIIx-luciferase together with either pcDNA-3, IKKβ or theD738 mutants described above (A). After 48 hours, the cells were lysedand luciferase activity was determined as previously described.

FIG. 9 depicts the results of a mutational analysis of W739 and W741within the NBD of human IKKβ. (A) The tryptophan residues at positions739 and 741 of IKKβ were substituted with alanine, phenylalanine,tyrosine or arginine using PCR-mutagenesis. (B) COS cells weretransiently transfected with either vector alone (pcDNA-3,1-xpress),IKKβ, W739A, W739F or W739Y together with FLAG-tagged NEMO as shown.After 48 hours, the cells were lysed and complexes wereimmunoprecipitated (IP) using anti-FLAG (M2)-coupled agarose beads.Prior to immunoprecipitation a portion of each lystate (5%) was retainedfor analysis (pre-IP). Proteins in samples were separated by SDS-PAGE(10%) and analyzed by immunoblotting (IB) using antibodies recognizingeither FLAG (M2) or xpress. The upper two panels show xpress-tagged IKKβand the lower panel shows FLAG-tagged NEMO. (C and D) COS cells weretransiently transfected with the plasmids shown followed byimmunoprecipitation and immunoblot analysis as described in B. (C and D)Hela cells were transiently transfected with pBIIx-luciferase togetherwith the plasmids shown and after 48 hours luciferase activity inlysates was determined.

FIG. 10 depicts the results of a mutational analysis of S740 within theNBD of human IKKβ. The serine residues at position 740 of IKKβ wassubstituted with alanine or glutamic acid using PCR-mutagenesis. (B) COScells were transiently transfected with the plasmids shown followed byimmunoprecipitation and immunoblot analysis as described in FIG. 2B. (C)Hela cells were transiently transfected for 48 hours with eitherIKKβ-FLAG or S740E-FLAG then treated for the times shown with TNFα (1μg/ml). Following lysis, complexes were precipitated using anti-FLAG(M2)-coupled agarose beads and an immune-complex kinase assay wasperformed using GST-IκBα (1-90) as a substrate as previously described.

FIG. 11 depicts the results of a mutational analysis of the IKKα NBD.(A) Each of the residues that comprise the NBD of IKKα (L738 to L743)were substituted with alanine by PCR-mutagenesis. COS cells weretransiently transfected with NEMO-FLAG together with either vector alone(pcDNA-3.1-xpress) or xpress-tagged versions of IKKα and the NBD mutantsas shown. Immunoprecipitation and immunoblot analysis of the IKKα-NEMOcomplexes was performed as described in FIG. 2B. (B) Hela cells weretransiently transfected with pBIIx-luciferase together with the plasmidsshown and after 48 hours luciferase activity in lysates was determined.

FIG. 12 depicts the results of an experiment demonstrating that apeptide encompassing the IKKβ NBD prevents the interaction of IKKα withNEMO. (A) Sequences of the NBD wild type and scrambled control peptides.The wild type peptide corresponds to residues 734 to 744 of IKKβ. (B)GST pull-down analysis was performed using GST-NEMO and in vitrotranscribed and translated IKKα (upper panel) and IKKβ (middle panel) inthe presence or absence of either vehicle (2% DMSO), scrambled or wildtype NBD peptide (500 and 1000 μM of each peptide). The lower panelshows a coomassie blue-stained gel demonstrating that neither peptideaffects the interaction of GST-NEMO with the glutathione-agarose beadsused for precipitation. (C) Densitometric analysis of autoradiographbands obtained following GST pull-down of IKKa and IKKb using GST-NEMOin the presence of a range of concentrations of wild type NBD peptide.The inset shows a representative experiment. The data are presented asthe pixel density as a percentage of control (no peptide) and representmeans±sd (n=11). Analysis was performed using the NIH-Image software.

DETAILED DESCRIPTION OF THE INVENTION I. General Description

The present invention provides anti-inflammatory compounds,pharmaceutical compositions thereof, and methods of use thereof fortreating inflammatory disorders. The present invention is based, atleast in part, on the identification of the NEMO binding domain (NBD) onIκB kinase-α (IKKα) and on IκB kinase-β (IKKβ).

Without intending to be limited by mechanism, it is believed that theanti-inflammatory compounds of the present invention act by blocking theinteraction of NEMO with an IKK (e.g., IKKβ or IKKα) at the NEMO bindingdomain (NBD), thereby inhibiting phosphorylation, degradation andsubsequent dissociation of IκB from NF-κB. This inhibition results inblockade of NF-κB activation associated with pro-inflammatory responses.

The present invention also provides methods for screening andidentifying anti-inflammatory compounds.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains.

As used herein, the term “binding” refers to the adherence of moleculesto one another, such as, but not limited to, enzymes to substrates,antibodies to antigens, DNA strands to their complementary strands.Binding occurs because the shape and chemical nature of parts of themolecule surfaces are “complementary”. A common metaphor is the“lock-and-key” used to describe how enzymes fit around their substrate.

The term “fusion peptide” or “fusion polypeptide” or “fusion protein”includes a peptide, polypeptide or protein that is obtained by combiningtwo distinct amino acid sequences. Typically, a partial sequence fromone peptide, polypeptide or protein is linked to another heterologouspeptide, polypeptide or protein, using art known techniques.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains areknown in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), β-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Non-limiting examples ofconservative substitutions that can be made in the anti-inflammatorycompounds of the present invention include substitution ofD-phenylalanine with D-tyrosine, D-pyridylalanine orD-homophenylalanine, substitution of D-leucine with D-valine or othernatural or non-natural amino acid having an aliphatic side chain and/orsubstitution of D-valine with D-leucine or other natural or non-naturalamino acid having an aliphatic side chain.

As used herein, the term “domain” refers to a part of a molecule orstructure that shares common physicochemical features, such as, but notlimited to, hydrophobic, polar, globular and helical domains orproperties. Specific examples of binding domains include, but are notlimited to, DNA binding domains and ATP binding domains.

As used herein, the term “membrane translocation domain” refers to apeptide capable of permeating the membrane of a cell and which is usedto transport attached peptides into a cell in vivo. Membranetranslocation domains include, but are not limited to, the third helixof the antennapedia homeodomain protein and the HIV-1 protein Tat.Additional membrane translocation domains are known in the art andinclude those described in, for example, Derossi et al., (1994) J. Biol.Chem. 269, 10444-10450; Lindgren et al., (2000) Trends Pharmacol. Sci.21, 99-103; Ho et al., Cancer Research 61, 474-477 (2001); U.S. Pat. No.5,888,762; U.S. Pat. No. 6,015,787; U.S. Pat. No. 5,846,743; U.S. Pat.No. 5,747,641; U.S. Pat. No. 5,804,604; and Published PCT applicationsWO 98/52614, WO 00/29427 and WO 99/29721. The entire contents of each ofthe foregoing references are incorporated herein by reference.

As used herein, the term “IκB” (I kappa B) refers to any one of severalmembers of a family of structurally related inhibitory proteins thatfunction in the regulation of NF-κB induction.

As used herein, the term “IκB-kinase” or “IκB protein kinase” or“IκB-kinase complex” or “IκB protein kinase complex” or “IKK” refers toa kinase that phosphorylates IκBs.

As used herein, the term “IKKα” refers to the α subunit of an IκB-kinasecomplex. As used herein, the term “IKKβ” refers to the β subunit of anIκB-kinase complex.

As used herein, the term “NEMO” (NF-κB Essential Modulator), “IKKγ” or“IKKAP” refers to the protein which binds to IKKs and facilitates kinaseactivity.

As used herein, the term “NEMO Binding Domain” or “NBD” includes anydomain capable of binding to NEMO at the region where NEMO usuallyinteracts with an IKK (e.g., IKKα or IKKβ). NEMO binding domainsinclude, for example, the α2-region (residues 737-742) of wild-typeIKKβ, or the corresponding six amino acid sequence of wild-type IKKα(residues 738-743) which are critical for interaction with NEMO. Thenucleic acid sequence and the corresponding amino acid sequence of thewild-type IKKβNBD are provided in SEQ ID NO:1 (GenBank Accession No.AR067807; nucleotides 2203-2235) and SEQ ID NO:2, respectively.

The terms “analogue”, “derivative” and “mimetic” as used herein areintended to include molecules which mimic the chemical structure of apeptidic structure and retain the functional properties of the peptidicstructure. Approaches to designing peptide analogs, derivatives andmimetics are known in the art. For example, see Farmer, P. S. in DrugDesign (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp.119-143; Ball. J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55;Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; andFreidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270. See also Sawyer,T. K. (1995) “Peptidomimetic Design and Chemical Approaches to PeptideMetabolism” in Taylor, M. D. and Amidon, G. L. (eds.) Peptide-Based DrugDesign: Controlling Transport and Metabolism, Chapter 17; Smith, A. B.3rd, et al. (1995) J. Am. Chem. Soc. 117:11113-11123; Smith, A. B. 3rd,et al. (1994) J. Am. Chem. Soc. 116:9947-9962; and Hirschman, R., et al.(1993) J. Am. Chem. Soc. 115:12550-12568.

As used herein, a “derivative” of a compound X (e.g., a peptide or aminoacid) refers to a form of X in which one or more reaction groups on thecompound have been derivatized with a substituent group. Examples ofpeptide derivatives include peptides in which an amino acid side chain,the peptide backbone, or the amino- or carboxy-terminus has beenderivatized (e.g., peptidic compounds with methylated amide linkages).As used herein an “analogue” of a compound X refers to a compound whichretains chemical structures of X necessary for functional activity of Xyet which also contains certain chemical structures which differ from X.An examples of an analogue of a naturally-occurring peptide is a peptidewhich includes one or more non-naturally-occurring amino acids. As usedherein, a “mimetic” of a compound X refers to a compound in whichchemical structures of X necessary for functional activity of X havebeen replaced with other chemical structures which mimic theconformation of X. Examples of peptidomimetics include peptidiccompounds in which the peptide backbone is substituted with one or morebenzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science260:1937-1942).

The term mimetic, and in particular, peptidomimetic, is intended toinclude isosteres. The term “isostere” as used herein is intended toinclude a chemical structure that can be substituted for a secondchemical structure because the steric conformation of the firststructure fits a binding site specific for the second structure. Theterm specifically includes peptide back-bone modifications (i.e., amidebond mimetics) well known to those skilled in the art. Suchmodifications include modifications of the amide nitrogen, the α-carbon,amide carbonyl, complete replacement of the amide bond, extensions,deletions or backbone crosslinks. Several peptide backbone modificationsare known, including ψ[CH₂S], ψ[CH₂NH], ψ[CSNH₂], ψ[NHCO], ψ[COCH₂], andψ[(E) or (Z) CH═CH]. In the nomenclature used above, ψ indicates theabsence of an amide bond. The structure that replaces the amide group isspecified within the brackets.

Other possible modifications include an N-alkyl (or aryl) substitution(ψ[CONR]), or backbone crosslinking to construct lactams and othercyclic structures. Other derivatives of the anti-inflammatory compoundsof the invention include C-terminal hydroxymethyl derivatives,O-modified derivatives (e.g., C-terminal hydroxymethyl benzyl ether),N-terminally modified derivatives including substituted amides such asalkylamides and hydrazides and anti-inflammatory compounds in which aC-terminal phenylalanine residue is replaced with a phenethylamideanalogue (e.g., Val-Phe-phenethylamide as an analogue of the tripeptideVal-Phe-Phe).

As used herein, the term “wild-type” refers to the genotype andphenotype that is characteristic of most of the members of a speciesoccurring naturally and contrasting with the genotype and phenotype of amutant.

III. Specific Embodiments A. Anti-Inflammatory Compounds

The present invention provides anti-inflammatory compounds comprising aNEMO binding domain (NBD). Any molecule comprising a domain that iscapable of binding to NEMO at the region where NEMO usually interactswith an IKK (e.g., IKKα or IKKβ) may be used to prepare theanti-inflammatory compounds of the present invention. Examples of suchmolecules include peptides comprising D- and/or L-configuration aminoacids; derivatives, analogues, and mimetics of peptidic compounds;antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic,chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fabexpression library fragments, and epitope-binding fragments ofantibodies); and small organic and inorganic molecules (e.g., moleculesobtained from combinatorial and natural product libraries).

In a preferred embodiment, the anti-inflammatory compounds of theinvention comprise fusions of a NEMO binding domain and at least onemembrane translocation domain which facilitates membrane translocationof the anti-inflammatory compounds of the invention in vivo.

Anti-inflammatory compounds of the present invention may be designedbased on the wild type amino acid sequence of the NBD of IKKα or IKKβ(SEQ ID NO:2). Any fragment of the wild type amino acid sequence of theNBD of IKKα or IKKβ capable of binding NEMO may be used to prepare ananti-inflammatory compound of this invention. Point mutations,insertions, or deletions of these wild type sequences (using the methodsdescribed herein) may be used to generate additional anti-inflammatorycompounds. Peptides containing conservative amino acid substitutions atpositions 737, 740 and 742 of the peptide set forth in SEQ ID NO:2 areparticularly useful anti-inflammatory compounds of the invention (seeTable 1 for examples of conservative substitutions which have nosignificant effect on the ability of the peptides to bind NEMO). Inaddition, naturally occurring allelic variants of the IKKβ gene thatretain the ability to bind NEMO may be used to prepare anti-inflammatorycompounds.

In one embodiment, the anti-inflammatory compounds of the presentinvention comprise: (a) peptides which include, or consist of, the aminoacid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or 19; (b) a peptide fragment of at least three aminoacids of the amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 or 19; (c) peptides which include aconservative amino acid substitution of the amino acid sequences of SEQID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19;and (d) naturally occurring amino acid sequence variants of the aminoacid sequences of SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or 19.

The anti-inflammatory compounds of the present invention may alsoinclude NEMO-specific receptors, such as somatically recombined peptidereceptors like specific antibodies or T-cell antigen receptors (seeHarlow & Lane, (1988) Antibodies—A Laboratory Manual, Cold Spring HarborLaboratory Press) and other natural intracellular binding agentsidentified with assays such as one, two and three-hybrid screens,non-natural intracellular binding agents identified in screens ofchemical libraries such as described below.

The anti-inflammatory compounds of the present invention are capable ofdown-regulating NEMO. Down-regulation is defined herein as a decrease inactivation, function or synthesis of NEMO, its ligands or activators. Itis further defined to include an increase in the degradation of the NEMOgene, its protein product, ligands or activators. Down-regulation may beachieved in a number of ways, for example, by destabilizing the bindingof NEMO to an IKK (e.g., IKKβ or IKKα); or by blocking thephosphorylation of IκB and causing the subsequent degradation of thisprotein.

Phosphorylation of IκB by IKKβ results in ubiquitination and degradationof IκB and subsequent dissociation of IκB, allowing for nucleartranslocation of NF-κB, leading to up-regulation of genes critical tothe inflammatory response. The anti-inflammatory compounds of thepresent invention may therefore be used to down-regulate NF-κB function.Down-regulation of NF-κB may also be accomplished by the use ofanti-inflammatory compounds comprising polyclonal or monoclonalantibodies or fragments thereof directed against a NBD or NEMO itself.This invention further includes small molecules having thethree-dimensional structure necessary to bind with sufficient affinityto a NBD or NEMO itself to, e.g., block NEMO interactions with IKKβ.IKKβblockade resulting in decreased degradation of IκB and decreasedactivation of NF-κB make these small molecules useful as therapeuticagents in treating or preventing inflammation.

In one embodiment, the present invention provides an anti-inflammatorycompound of the formulaX_(a)—X_(b),where X_(a) is a membrane translocation domain comprising from 6 to 15amino acid residues; and X_(b) is a NEMO binding sequence. The compoundcan, optionally, include a modifying group at the N-terminus, theC-terminus or both.

X_(b) is a NEMO binding sequence comprising from 6 to 9 amino acidresidues. In one embodiment, X_(b) consists of the following structure(Y)_(n)—X₁—X₂—X₃—X₄—X₅—X₆-(A)_(m)where n and m are each, independently, 0 or 1 and A and Y each comprisesfrom 1 to about 3 amino acid residues. When n is 1, Y is, preferably thesequence TA. When m is 1, A is preferably the sequence QTE. X₁ is L, A,I or nor-leucine (Nle); X₂ is D, E, N, Q, homoserine (Hser) or2-ketopropylalanine (2-ketopropy-A); X₃ is W, F Y, 4-biphenyl-alanine(Bpa), homophenylalanine (Hphe), 2-Naphthylalanine (2-NaI),1-Naphthylalanine (1-NaI), or cycloxexyl-alanine (Cha); X₄ is S, A, E,L, T, nor-leucine (Nle), or homoserine (Hser); X₅ is W, H,homophenylalanine (Hphe), 2-Naphthylalanine (2-NaI), 1-Naphthylalanine(1-NaI), O-benzyl serine (SeroBn), or 3-Pyridylalanine (3-Pal); and X₆is L, A, I, or nor-leucine (Nle).

Preferably, X_(b) is a sequence selected from among TALDWSWLQTE (SEQ IDNO:28); LDWSWLQTE (SEQ ID NO:29); TALDWSWL (SEQ ID NO:30); ALDWSWLQTE(SEQ ID NO:31); LDWSWLQTE (SEQ ID NO:32); LDWSWL (SEQ ID NO:33);TALDWSWLQT (SEQ ID NO:34); TALDWSWLQ (SEQ ID NO:35); ALDWSWLQT (SEQ IDNO:36); LDWSWLQ (SEQ ID NO:37); LDWSWLQT (SEQ ID NO:38); ADWSWL (SEQ IDNO:39); LDWSWA (SEQ ID NO:40); ADWSWA (SEQ ID NO:41); LDFSWL (SEQ IDNO:42); LDYSWL (SEQ ID NO:43); LDWAWL (SEQ ID NO:44); LDWEWL (SEQ IDNO:45); TAADWSWLQTE (SEQ ID NO:46); ADWSWLQTE (SEQ ID NO:47); TAADWSWL(SEQ ID NO:48); AADWSWLQTE (SEQ ID NO:49); ADWSWLQTE (SEQ ID NO:50);ADWSWL (SEQ ID NO:51); TAADWSWLQT (SEQ ID NO:52); TAADWSWLQ (SEQ IDNO:53); AADWSWLQT (SEQ ID NO:54); ADWSWLQ (SEQ ID NO:55); ADWSWLQT (SEQID NO:56); ALDWSWAQTE (SEQ ID NO:57); LDWSWAQTE (SEQ ID NO:58); TALDWSWA(SEQ ID NO:59); ALDWSWAQTE (SEQ ID NO:60); LDWSWAQTE (SEQ ID NO:61);LDWSWA (SEQ ID NO:62); TALDWSWAQT (SEQ ID NO:63); TALDWSWAQ (SEQ IDNO:64); ALDWSWAQT (SEQ ID NO:65); LDWSWAQ (SEQ ID NO:66); LDWSWAQT (SEQID NO:67); TAADWSWAQTE (SEQ ID NO:68); ADWSWAQTE (SEQ ID NO:69);TAADWSWA (SEQ ID NO:70); AADWSWAQTE (SEQ ID NO:71); ADWSWAQTE (SEQ IDNO:72); ADWSWA (SEQ ID NO:73); TAADWSWAQT (SEQ ID NO:74); TAADWSWAQ (SEQID NO:75); AADWSWAQT (SEQ ID NO:76); ADWSWAQ (SEQ ID NO:77); ADWSWAQT(SEQ ID NO:78); TALDFSWLQTE (SEQ ID NO:79); LDFSWLQTE (SEQ ID NO:80);TALDFSWL (SEQ ID NO:81); ALDFSWLQTE (SEQ ID NO:82); LDFSWLQTE (SEQ IDNO:83); LDFSWL (SEQ ID NO:84); TALDFSWLQT (SEQ ID NO:85); TALDFSWLQ (SEQID NO:86); ALDFSWLQT (SEQ ID NO:87); LDFSWLQ (SEQ ID NO:88); LDFSWLQT(SEQ ID NO:89); TALDYSWLQTE (SEQ ID NO:90); LDYSWLQTE (SEQ ID NO:91);TALDYSWL (SEQ ID NO:92); ALDYSWLQTE (SEQ ID NO:93); LDYSWLQTE (SEQ IDNO:94); LDYSWL (SEQ ID NO:95); TALDYSWLQT (SEQ ID NO:96); TALDYSWLQ (SEQID NO:97); ALDYSWLQT (SEQ ID NO:98); LDYSWLQ (SEQ ID NO:99); LDYSWLQT(SEQ ID NO:100); TALDWAWLQTE (SEQ ID NO:101); LDWAWLQTE (SEQ ID NO:102);TALDWAWL (SEQ ID NO:103); ALDWAWLQTE (SEQ ID NO:104); LDWAWLQTE (SEQ IDNO:105); LDWAWL (SEQ ID NO:106); TALDWAWLQT (SEQ ID NO:107); TALDWAWLQ(SEQ ID NO:108); ALDWAWLQT (SEQ ID NO:109); LDWAWLQ (SEQ ID NO:110);LDWAWLQT (SEQ ID NO:111); TALDWEWLQTE (SEQ ID NO:112); LDWEWLQTE (SEQ IDNO:113); TALDWEWL (SEQ ID NO:114); ALDWEWLQTE (SEQ ID NO:115); LDWEWLQTE(SEQ ID NO:116); LDWEWL (SEQ ID NO:117); TALDWEWLQT (SEQ ID NO:118);TALDWEWLQ (SEQ ID NO:119); ALDWEWLQT (SEQ ID NO:120); LDWEWLQ (SEQ IDNO:121); and LDWEWLQT (SEQ ID NO:122).

X_(a) is a membrane transduction domain consisting of 6-15 amino acidresidues, preferably 6-12, or 6-10 amino acid residues. Preferably,X_(a) is a membrane translocation domain which comprises at least fivebasic amino acid residues, preferably at least five residuesindependently selected from L-arginine, D-arginine, L-lysine andD-lysine. Suitable membrane transduction domains include those disclosedherein.

In one embodiment, X_(a) is selected from among the amino acid sequencesRRMKWKK (SEQ ID NO: 123); YGRKKRRQRRR (SEQ ID NO: 124); ygrkkrrqrrr (SEQID NO: 125); YARKARRQARR (SEQ ID NO:126); yarkarrqarr (SEQ ID NO: 127);YARAARRAARR (SEQ ID NO: 128); yaraarraarr (SEQ ID NO: 129); rrmkwkk (SEQID NO: 130); (R)_(y) and (r)_(y), where y is 6 to 11. Lower case lettersindicate D-amino acid residues and upper case letters indicate L-aminoacid residues.

Examples of suitable peptides X_(a)—X_(b) include those having thefollowing sequences: RRMKWKKTALDWSWLQTE (SEQ ID NO: 131);rrmkwkkTALDWSWLQTE (SEQ ID NO:132); YGRKKRRQRRRTALDWSWLQTE (SEQ ID NO:133); ygrkkrrqrrrTALDWSWLQTE (SEQ ID NO: 134); rmmrTALDWSWLQTE (SEQ IDNO:135); RRRRRRRTALDWSWLQTE (SEQ ID NO:136); YARKARRQARRTALDWSWLQTE (SEQID NO:137); yarkarrqarrTALDWSWLQTE (SEQ ID NO:138);YARAARRAARRTALDWSWLQTE (SEQ ID NO: 139); yaraarraarrTALDWSWLQTE (SEQ IDNO: 140) YGRKKRRQRRRLDWSWL (SEQ ID NO:141); ygrkkrrqrrrLDWSWL (SEQ IDNO:142); RRMKWKKLDWSWL (SEQ ID NO: 143); rrmkwkkLDWSWL (SEQ ID NO: 144);mmrrrLDWSWL (SEQ ID NO: 145); YARAARRAARRLDWSWL (SEQ ID NO: 146);yaraarraarrLDWSWL (SEQ ID NO: 147); and RRRRRRRLDWSWL (SEQ ID NO: 148).

The anti-inflammatory compounds of the invention can optionally includemodifying groups attached to the C-terminus, the N-terminus or both. Forexample, suitable modifying groups which can be attached to theC-terminus include substituted and unsubstituted amino groups, forexample, —NH₂, —NH(alkyl) and —N(alkyl)₂ groups; and alkoxy groups, suchas linear, branched or cyclic C₁-C₆-alkoxy groups. A preferredC-terminal modifying group is the —NH₂ group. Suitable modifying groupswhich can be attached to the N-terminus include acyl groups, such as theacetyl group; and alkyl groups, preferably C₁-C₆-alkyl groups, morepreferably methyl.

In the anti-inflammatory compounds of the present invention the membranetranslocation domain, X_(a), may be present at the amino-terminus of thecompound and the NEMO binding sequence, X_(b), may be present at thecarboxyl-terminus of the compound (X_(a)—X_(b)). Alternatively, in theanti-inflammatory compounds of the present invention the membranetranslocation domain, X_(a), may be present at the carboxyl-terminus ofthe compound and the NEMO binding sequence, X_(b), may be present at theamino-terminus of the compound (X_(b)—X_(a)).

Particular anti-inflammatory compounds of the invention include thoselisted below:

Cmpd no.  1 H-RRMKWKKTALDWSWLQTE-NH₂  2 H-YGRKKRRQRRRTALDWSWLQTE-NH₂  3H-rrrrrrrTALDWSWLQTE-NH₂  4 H-YARKARRQARRTALDWSWLQTE-NH₂  5H-YARAARRAARRTALDWSWLQTE-NH₂  6 H-RRMKWKKLDWSWL-NH₂  7H-rrmkwkkLDWSWL-NH₂  8 H-rrrrrrrLDWSWL-NH₂  9 H-YARAARRAARRLDWSWL-NH₂ 10H-yaraarraarrLDWSWL-NH₂ 11 H-YGRKKRRQRRRLDWSWL-NH₂

B. Screening Assays

In addition, this invention also provides screening methods foridentifying anti-inflammatory compounds. The anti-inflammatory compoundsmay block the function, prevent the synthesis or reduce the biologicstability of IKKβ by interacting at the NBD of this molecule. Biologicstability is a measure of the time between the synthesis of the moleculeand its degradation. For example, the stability of a protein, peptide orpeptide mimetic (Kauvar, Nature Biotech. (1996) 14, 709) therapeutic maybe shortened by altering its sequence to make it more susceptible toenzymatic degradation.

The present invention also includes methods of screening for compoundswhich deactivate, or act as antagonists of IKKβ function. Such compoundsmay be useful in the modulation of pathological conditions associatedwith alterations in IKKβ or NF-κB protein levels.

The present invention also provides methods for isolating andidentifying binding partners of proteins of the invention, for example,compounds which interact with IKKβat the NBD of this molecule, orinteract with NEMO, thereby blocking NEMO interaction with IKKβ. Aprotein of the invention is mixed with a potential binding partner or anextract or fraction of a cell under conditions that allow for theassociation of potential binding partners with the proteins of theinvention. After mixing, peptides, polypeptides, proteins or othermolecules that have become associated with a protein of the inventionare separated from the mixture. The binding partner bound to the proteinof the invention can then be removed and further analyzed. To identifyand isolate a binding partner, the entire protein, for instance theentire IKKβ peptide can be used. Alternatively, a fragment of theprotein can be used. For example, the peptide fragment comprising NBDcan be used to block interaction of IKKβ with NEMO.

A variety of methods can be used to obtain an extract of a cell. Cellscan be disrupted using either physical or chemical disruption methods.Examples of physical disruption methods include, but are not limited to,sonication and mechanical shearing. Examples of chemical lysis methodsinclude, but are not limited to, detergent lysis and enzyme lysis. Askilled artisan can readily adapt methods for preparing cellularextracts in order to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with eitherIKKβ or NEMO under conditions in which association of the protein withthe binding partner can occur. A variety of conditions can be used, themost preferred being conditions that closely resemble conditions foundin the cytoplasm of a human cell. Features such as osmolarity, pH,temperature, and the concentration of cellular extract used, can bevaried to optimize the association of the protein with the bindingpartner.

After mixing under the appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be utilized toseparate the mixture. For example, antibodies specific to a protein ofthe invention can be used to immunoprecipitate the binding partnercomplex. Alternatively, standard chemical separation techniques such aschromatography and density-sediment centrifugation can be used.

After removal of non-associated cellular constituents found in theextract, the binding partner can be dissociated from the complex usingconventional methods. For example, dissociation can be accomplished byaltering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixedextract, the protein can be immobilized on a solid support. For example,the protein can be attached to a nitrocellulose matrix or acrylic beads.Attachment of the protein to a solid support aids in separatingpeptide-binding partner pairs from other constituents found in theextract. The identified binding partners can be either a single proteinor a complex made up of two or more proteins. Alternatively, bindingpartners may be identified using a Far-Western assay according to theprocedures of Takayama et al., (1997) Methods Mol. Biol. 69, 171-184 orSauder et al., (1996) J. Gen. Virol. 77, 991-996 or identified throughthe use of epitope tagged proteins or GST fusion proteins.

Alternatively, the nucleic acid molecules encoding the peptides of theinvention can be used in a yeast two-hybrid system. The yeast two-hybridsystem has been used to identify other protein partner pairs and canreadily be adapted to employ the nucleic acid molecules herein described(see, for example, Stratagene Hybrizap® two-hybrid system).

Another embodiment of the present invention provides methods foridentifying agents that modulate at least one activity of NEMO or IKKβ.Such methods or assays may utilize any means of monitoring or detectingthe desired activity.

In one format, the relative amounts of a protein of the inventionbetween a cell population that has been exposed to the agent to betested compared to an un-exposed control cell population may be assayed.In this format, probes such as specific antibodies are used to monitorthe differential expression of the protein in the different cellpopulations. Cell lines or populations are exposed to the agent to betested under appropriate conditions and time. Cellular lysates may beprepared from the exposed cell line or population and a control,unexposed cell line or population. The cellular lysates are thenanalyzed with the probe.

Antibody probes are prepared by immunizing suitable mammalian hosts inappropriate immunization protocols using the peptides. Peptides orproteins comprising the NBD are of sufficient length, or if desired, asrequired to enhance immunogenicity, conjugated to suitable carriers.Methods for preparing immunogenic conjugates with carriers such as BSA,KLH, or other carrier proteins are well known in the art. In somecircumstances, direct conjugation using, for example, carbodiimidereagents may be effective; in other instances linking reagents such asthose supplied by Pierce Chemical Co. may be desirable to provideaccessibility to the hapten. The hapten peptides can be extended ateither the amino or carboxy terminus with a cysteine residue orinterspersed with cysteine residues, for example, to facilitate linkingto a carrier. Administration of the immunogens is conducted generally byinjection over a suitable time period and with use of suitableadjuvants, as is generally understood in the art. During theimmunization schedule, titers of antibodies are taken to determineadequacy of antibody formation. While the polyclonal antisera producedin this way may be satisfactory for some applications, forpharmaceutical compositions, use of monoclonal preparations ispreferred. Immortalized cell lines which secrete the desired monoclonalantibodies may be prepared using the standard method of Kohler &Milstein, (1992) Biotechnology 24, 524-526 or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the peptide hapten,peptide or protein.

When the appropriate immortalized cell culture secreting the desiredantibody is identified, the cells can be cultured either in vitro or byproduction in ascites fluid. The desired monoclonal antibodies may berecovered from the culture supernatant or from the ascites supernatant.Fragments of the monoclonals or the polyclonal antisera which containthe immunologically significant portion can be used as antagonists, aswell as the intact antibodies. Use of immunologically reactivefragments, such as the Fab, Fab′ of F(ab′)2 fragments is oftenpreferable, especially in a therapeutic context, as these fragments aregenerally less immunogenic than the whole immunoglobulin.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Antibody regions that bindspecifically to the desired regions of the protein can also be producedin the context of chimeras with multiple species origin. Agents that areassayed in the above method can be randomly selected or rationallyselected or designed. As used herein, an agent is said to be randomlyselected when the agent is chosen randomly without considering thespecific sequences involved in the association of the a protein of theinvention alone or with its associated substrates, binding partners,etc. An example of randomly selected agents is the use a chemicallibrary or a peptide combinatorial library, or a growth broth of anorganism.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a non-random basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. Agents can be rationally selected or rationallydesigned by utilizing the peptide sequences that comprises the NBD onIKKβ or the IKKβ binding domain on NEMO. For example, a rationallyselected peptide agent can be a peptide whose amino acid sequence isidentical to the amino acid sequence of SEQ ID NO: 2 or a peptide withconservative substitutions thereof.

The compounds of the present invention can be, as examples, peptides,small molecules, vitamin derivatives, as well as carbohydrates. Askilled artisan can readily recognize that there is no limit as to thestructural nature of the compounds of the present invention.

The peptide compounds of the invention can be prepared using standardsolid phase (or solution phase) peptide synthesis methods, as is knownin the art. In addition, the DNA encoding these peptides may besynthesized using commercially available oligonucleotide synthesisinstrumentation and produced recombinantly using standard recombinantproduction systems. The production using solid phase peptide synthesisis necessitated if non-gene-encoded amino acids are to be included.

The present invention further provides isolated nucleic acid moleculesthat encode the peptide having a NBD and conservative nucleotidesubstitutions thereof, preferably in isolated form. Conservativenucleotide substitutions include nucleotide substitutions which do noteffect the coding for a particular amino acid as most amino acids havemore than one codon (see King & Stansfield (Editors), A Dictionary ofGenetics, Oxford University Press, 1997 at page 19). Conservativenucleotide substitutions therefore also include silent mutations anddifferential codon usage. For example, the invention includes thenucleic acid molecule set forth in SEQ ID NO: 1, which encodes thepeptide set forth in SEQ ID NO:2, and conservative nucleotidesubstitutions thereof. The invention also includes nucleic acidsencoding the peptides set forth in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18 and 19 and conservative nucleotidesubstitutions thereof. Any nucleic acid that encodes the peptides setforth in SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18 and 19 is encompassed by the invention, given the multiplepermutations of nucleotide sequences possible which encode thesepeptides.

Specific examples of nucleic acids encompassed by this inventioninclude, but are not limited to the following: (1) the amino acids ofthe peptide of SEQ ID NO:2 can be encoded by the nucleic acid sequenceTTAGATTGGTCTTGGTTA (SEQ ID NO:24) or TTGGACTGGTCCTGGCTA (SEQ ID NO:25);and (2) the amino acids of the peptide of SEQ ID NO:15 can be encoded bythe nucleic acid sequence TTAGATTGGTCTTATCTG (SEQ ID NO: 26) orCTTGACTGGTCATACTTA (SEQ ID NO: 27).

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid encoding other polypeptides from the source of nucleicacid. Modifications to the primary structure of the nucleic acid itselfby deletion, addition, or alteration of the amino acids incorporatedinto the protein sequence during translation can be made withoutdestroying the activity of the peptide. Such substitutions or otheralterations result in peptide having an amino acid sequence encoded by anucleic acid falling within the contemplated scope of the presentinvention.

Another class of compounds of the present invention are antibodiesimmunoreactive with critical positions of proteins of the invention.Antibody agents are obtained by immunization of suitable mammaliansubjects with peptides, containing as antigenic regions, those portionsof the protein intended to be targeted by the antibodies.

C. High Throughput Assays

Introduction—The power of high throughput screening is utilized to thesearch for new anti-inflammatory compounds which are capable ofinteracting with NEMO. For general information on high-throughputscreening, see, for example, Cost-Effective Strategies for Automated andAccelerated High-Throughput Screening, IBCS Biomedical Library Series,IBC United States Conferences, 1996; Devlin (Editor), High ThroughputScreening, Marcel Dekker 1998; U.S. Pat. No. 5,763,263. High throughputassays utilize one or more different assay techniques.Immunodiagnostics and Immunoassays—These are a group of techniques usedfor the measurement of specific biochemical substances, commonly at lowconcentrations in complex mixtures such as biological fluids, thatdepend upon the specificity and high affinity shown by suitably preparedand selected antibodies for their complementary antigens. A substance tobe measured must, of necessity, be antigenic-either an immunogenicmacromolecule or a haptenic small molecule. To each sample a known,limited amount of specific antibody is added and the fraction of theantigen combining with it, often expressed as the bound:free ratio, isestimated, using as indicator a form of the antigen labeled withradioisotope (radioimmunoassay), fluorescent molecule(fluoroimmunoassay), stable free radical (spin immunoassay), enzyme(enzyme immunoassay), or other readily distinguishable label.

Antibodies can be labeled in various ways, including: enzyme-linkedimmunosorbent assay (ELISA); radioimmuno assay (RIA); fluorescentimmunoassay (FIA); chemiluminescent immunoassay (CLIA); and labeling theantibody with colloidal gold particles (immunogold).

Common assay formats include the sandwich assay, competitive orcompetition assay, latex agglutination assay, homogeneous assay,microtitre plate format and the microparticle-based assay.

Enzyme-linked immunosorbent assay (ELISA)—ELISA is an immunochemicaltechnique that avoids the hazards of radiochemicals and the expense offluorescence detection systems. Instead, the assay uses enzymes asindicators. ELISA is a form of quantitative immunoassay based on the useof antibodies (or antigens) that are linked to an insoluble carriersurface, which is then used to “capture” the relevant antigen (orantibody) in the test solution. The antigen-antibody complex is thendetected by measuring the activity of an appropriate enzyme that hadpreviously been covalently attached to the antigen (or antibody).

For information on ELISA techniques, see, for example, Crowther, ELISA:Theory and Practice (Methods in Molecular Biology, Vol. 42), HumanaPress, 1995; Challacombe & Kemeny, ELISA and Other Solid PhaseImmunoassays: Theoretical and Practical Aspects, John Wiley, 1998;Kemeny, A Practical Guide to ELISA, Pergamon Press, 1991; Ishikawa,Ultrasensitive and Rapid Enzyme Immunoassay (Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 27), Elsevier, 1991.

Colorimetric Assays for Enzymes—Colorimetry is any method ofquantitative chemical analysis in which the concentration or amount of acompound is determined by comparing the color produced by the reactionof a reagent with both standard and test amounts of the compound, oftenusing a calorimeter. A colorimeter is a device for measuring colorintensity or differences in color intensity, either visually orphotoelectrically.

Standard calorimetric assays of beta-galactosidase enzymatic activityare well known to those skilled in the art (see, for example, Norton etal., (1985) Mol. Cell. Biol. 5, 281-290). A calorimetric assay can beperformed on whole cell lysates usingO-nitrophenyl-beta-D-galactopyranoside (ONPG, Sigma) as the substrate ina standard calorimetric beta-galactosidase assay (Sambrook et al.,(1989) Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Press).Automated calorimetric assays are also available for the detection ofbeta-galactosidase activity, as described in U.S. Pat. No. 5,733,720.

Immunofluorescence Assays—Immunofluorescence or immunofluorescencemicroscopy is a technique in which an antigen or antibody is madefluorescent by conjugation to a fluorescent dye and then allowed toreact with the complementary antibody or antigen in a tissue section orsmear. The location of the antigen or antibody can then be determined byobserving the fluorescence by microscopy under ultraviolet light.

For general information on immunofluorescent techniques, see, forexample, Knapp et al., (1978) Immunofluorescence and Related StainingTechniques, Elsevier; Allan, (1999) Protein Localization by FluorescentMicroscopy: A Practical Approach (The Practical Approach Series, Vol.218) Oxford University Press; Beutner, (1983) Defined Immunofluorescenceand Related Cytochemical Methods, New York Academy of Sciences; Caul,(1993) Immunofluorescence Antigen Detection Techniques in DiagnosticMicrobiology, Cambridge University Press. For detailed explanations ofimmunofluorescent techniques applicable to the present invention, see,U.S. Pat. Nos. 5,912,176; 5,869,264; 5,866,319; and 5,861,259.

Biochips—The peptides of the invention can be used on an array ormicroarray for high-throughput screening for agents which interact witheither the nucleic acids of the invention or their correspondingproteins.

An “array” or “microarray” generally refers to a grid system which haseach position or probe cell occupied by a defined nucleic acid fragmentsalso known as oligonucleotides. The arrays themselves are sometimesreferred to as “chips” or “biochips” which are high-density nucleic acidand peptide microarrays often having thousands of probe cells in avariety of grid styles.

A typical molecular detection chip includes a substrate on which anarray of recognition sites, binding sites or hybridization sites arearranged. Each site has a respective molecular receptor which binds orhybridizes with a molecule having a predetermined structure. The solidsupport substrates which can be used to form surface of the array orchip include organic and inorganic substrates, such as glass,polystyrenes, polyimides, silicon dioxide and silicon nitride. Fordirect attachment of probes to the electrodes, the electrode surfacemust be fabricated with materials capable of forming conjugates with theprobes.

Once the array is fabricated, a sample solution is applied to themolecular detection chip and molecules in the sample bind or hybridizeat one or more sites. The sites at which binding occurs are detected,and one or more molecular structures within the sample are subsequentlydeduced. Detection of labeled batches is a traditional detectionstrategy and includes radioisotope, fluorescent and biotin labels, butother options are available, including electronic signal transduction.

The methods of this invention will find particular use wherever highthrough-put of samples is required. In particular, this invention isuseful in ligand screening settings and for determining the compositionof complex mixtures.

Polypeptides are an exemplary system for exploring the relationshipbetween structure and function in biology. When the twenty naturallyoccurring amino acids are condensed into a polymeric molecule they forma wide variety of three-dimensional configurations, each resulting froma particular amino acid sequence and solvent condition. For example, thenumber of possible polypeptide configurations using the twenty naturallyoccurring amino acids for a polymer five amino acids long is over threemillion. Typical proteins are more than one-hundred amino acids inlength.

In typical applications, a complex solution containing one or moresubstances to be characterized contacts a polymer array comprisingpolypeptides. The polypeptides of the invention can be prepared byclassical methods known in the art, for example, by using standard solidphase techniques. The standard methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensation,classical solution synthesis and recombinant DNA technology (seeMerrifield, (1963) Am. Chem. Soc. 85, 2149-2152).

In a preferred embodiment, the polypeptides or proteins of the array canbind to other co-receptors to form a heteroduplex on the array. In yetanother embodiment, the polypeptides or proteins of the array can bindto peptides or small molecules.

D. Uses for the Anti-Inflammatory Compounds of the Present Invention

The anti-inflammatory compounds of the present invention (e.g.,compounds that modulate the expression of NEMO or compounds such asagonists or antagonists of at least one activity of NEMO) may be used tomodulate inflammation and treat or diagnose an inflammatory disorder ina subject. The methods include administering to a subject ananti-inflammatory compound of the invention in an amount effective totreat an inflammatory disorder.

As used herein, an “inflammatory disorder” is intended to include adisease or disorder characterized by, caused by, resulting from, orbecoming affected by inflammation. An inflammatory disorder may becaused by or be associated with biological and pathological processesassociated with NEMO or IKKβ function and activity and/or with NF-κBmediated processes. Examples of inflammatory diseases or disordersinclude, but not limited to, acute and chronic inflammation disorderssuch as asthma, psoriasis, rheumatoid arthritis, osteoarthritis,psoriatic arthritis, inflammatory bowel disease (Crohn's disease,ulcerative colitis), sepsis, vasculitis, and bursitis; autoimmunediseases such as Lupus, Polymyalgia, Rheumatica, Scleroderma, Wegener'sgranulomatosis, temporal arteritis, cryoglobulinemia, and multiplesclerosis; transplant rejection; osteoporosis; cancer, including solidtumors (e.g., lung, CNS, colon, kidney, and pancreas); Alzheimer'sdisease; atherosclerosis; viral (e.g., HIV or influenza) infections;chronic viral (e.g., Epstein-Barr, cytomegalovirus, herpes simplexvirus) infection; and ataxia telangiectasia.

Pathological processes refer to a category of biological processes whichproduce a deleterious effect. For example, unregulated expression ofNF-κB is associated with pro-inflammatory processes underlying certainpathological processes. As used herein, an anti-inflammatory compound issaid to modulate a pathological process when the compound reduces thedegree or severity of the process. For instance, pro-inflammatoryresponses may be prevented or pathological processes modulated by theadministration of anti-inflammatory compounds which reduce, promote ormodulate in some way the expression or at least one activity of NEMO orIKKβ.

The anti-inflammatory compounds of the present invention may, therefore,be used to treat diseases with an NF-κB inflammatory component. Suchdiseases include, but are not limited to, osteoporosis, rheumatoidarthritis, atherosclerosis, asthma (Ray & Cohn, (1999) J. Clin. Invest.104, 985-993; Christman et al., (2000) Chest 117, 1482-1487) andAlzheimer's disease. For a review of diseases with an NF-κB inflammatorycomponent, see Epstein, (1997) New Eng. J. Med. 336, 1066-1071; Lee etal., (1998) J. Clin. Pharmacol. 38, 981-993; Brand et al., (1997) Exp.Physiol. 82, 297-304.

Pathological processes associated with a pro-inflammatory response inwhich the anti-inflammatory compounds of the invention would be usefulfor treatment include, but are not limited to, asthma, allergies such asallergic rhinitis, uticaria, anaphylaxis, drug sensitivity, foodsensitivity and the like; cutaneous inflammation such as dermatitis,eczema, psorisis, contact dermatitis, sunburn, aging, and the like;arthritis such as osteoarthritis, psoriatic arthritis, lupus,spondylarthritis and the like. Anti-inflammatory compounds are alsouseful for treating chronic obstruction pulmonary disease and chronicinflammatory bowel disease. The anti-inflammatory compounds of thepresent invention may further be used to replace corticosteroids in anyapplication in which corticosteroids are used includingimmunosuppression in transplants and cancer therapy.

As used herein, the term “subject” includes warm-blooded animals,preferably mammals, including humans. In a preferred embodiment, thesubject is a primate. In an even more preferred embodiment, the primateis a human.

As used herein, the term “administering” to a subject includesdispensing, delivering or applying an anti-inflammatory compound, e.g.,an anti-inflammatory compound in a pharmaceutical formulation (asdescribed herein), to a subject by any suitable route for delivery ofthe compound to the desired location in the subject, including deliveryby either the parenteral or oral route, intramuscular injection,subcutaneous/intradermal injection, intravenous injection, buccaladministration, transdermal delivery and administration by the rectal,colonic, vaginal, intranasal or respiratory tract route (e.g., byinhalation).

As used herein, the term “effective amount” includes an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result, e.g., sufficient to treat an inflammatory disorder in asubject. An effective amount of an anti-inflammatory compound of theinvention, as defined herein may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of thecompound to elicit a desired response in the subject. Dosage regimensmay be adjusted to provide the optimum therapeutic response. Aneffective amount is also one in which any toxic or detrimental effects(e.g., side effects) of the compound are outweighed by thetherapeutically beneficial effects.

A therapeutically effective amount of an anti-inflammatory compound ofthe invention (i.e., an effective dosage) may range from about 0.001 to30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight,more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciatethat certain factors may influence the dosage required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of an anti-inflammatorycompound of the invention can include a single treatment or, preferably,can include a series of treatments. In one example, a subject is treatedwith an anti-inflammatory compound of the invention in the range ofbetween about 0.1 to 20 mg/kg body weight, one time per week for betweenabout 1 to 10 weeks, preferably between 2 to 8 weeks, more preferablybetween about 3 to 7 weeks, and even more preferably for about 4, 5, or6 weeks. It will also be appreciated that the effective dosage of ananti-inflammatory compound of the invention used for treatment mayincrease or decrease over the course of a particular treatment.

The anti-inflammatory compounds of the present invention can be providedalone, or in combination with other agents that modulate a particularpathological process. For example, an anti-inflammatory compound of thepresent invention can be administered in combination with other knownanti-inflammatory agents. Known anti-inflammatory agents that may beused in the methods of the invention can be found in Harrison'sPrinciples of Internal Medicine, Thirteenth Edition, Eds. T. R. Harrisonet al. McGraw-Hill N.Y., N.Y.; and the Physicians Desk Reference 50thEdition 1997, Oradell N.J., Medical Economics Co., the complete contentsof which are expressly incorporated herein by reference. Theanti-inflammatory compounds of the invention and the additionalanti-inflammatory agents may be administered to the subject in the samepharmaceutical composition or in different pharmaceutical compositions(at the same time or at different times).

The present invention further provides methods for modulating signaltransduction involving IκB in a cell. The methods include modulatingIKKβ activity, e.g. by contacting a cell with an anti-inflammatorycompound. The anti-inflammatory compound may, for example, inhibit theinteraction of NEMO with IKKβ at the NBD, thereby inhibiting IKKβfunction. The cell may reside in culture or in situ, i.e., within thenatural host.

For diagnostic uses, the anti-inflammatory compounds of the inventionmay be labeled, such as with fluorescent, radioactive, chemiluminescent,or other easily detectable molecules. The label may be conjugated eitherdirectly or indirectly to the anti-inflammatory compound.

E. Pharmaceutical Preparations

The invention also includes pharmaceutical compositions comprising theanti-inflammatory compounds of the invention together with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described inGennaro et al., (1995) Remington's Pharmaceutical Sciences, MackPublishing Company. In addition to the pharmacologically active agent,the compositions of the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically for delivery to the siteof action. Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and dextran.Optionally, the suspension may also contain stabilizers. Liposomes canalso be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulations may be usedsimultaneously to achieve systemic administration of the activeingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

The anti-inflammatory compounds of the invention can also beincorporated into pharmaceutical compositions which allow for thesustained delivery of the anti-inflammatory compounds to a subject for aperiod of at least several weeks to a month or more. Such formulationsare described in U.S. Pat. Nos. 5,968,895 and 6,180,608 B1, the contentsof each of which are incorporated herein by reference.

The anti-inflammatory compounds of the present invention may beadministered via parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal or buccal routes. Alternatively, orconcurrently, administration may be by the oral route or by inhalationor lavage, directly to the lungs. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

The anti-inflammatory compounds used in the methods of treatmentdescribed herein may be administered systemically or topically,depending on such considerations as the condition to be treated, needfor site-specific treatment, quantity of drug to be administered andsimilar considerations.

Topical administration may be used. Any common topical formation such asa solution, suspension, gel, ointment or salve and the like may beemployed. Preparation of such topical formulations are well described inthe art of pharmaceutical formulations as exemplified, for example, byRemington's Pharmaceutical Sciences. For topical application, thesecompounds could also be administered as a powder or spray, particularlyin aerosol form. The active ingredient may be administered inpharmaceutical compositions adapted for systemic administration. As isknown, if a drug is to be administered systemically, it may be confectedas a powder, pill, tablet or the like or as a syrup or elixir for oraladministration. For intravenous, intraperitoneal or intra-lesionaladministration, the compound will be prepared as a solution orsuspension capable of being administered by injection. In certain cases,it may be useful to formulate these compounds in suppository form or asan extended release formulation for deposit under the skin orintramuscular injection. In a preferred embodiment, theanti-inflammatory compounds of the invention may be administered byinhalation. For inhalation therapy the compound may be in a solutionuseful for administration by metered dose inhalers or in a form suitablefor a dry powder inhaler.

An effective amount is that amount which will modulate the activity oralter the level of a target protein. A given effective amount will varyfrom condition to condition and in certain instances may vary with theseverity of the condition being treated and the patient's susceptibilityto treatment. Accordingly, a given effective amount will be bestdetermined at the time and place through routine experimentation.However, it is anticipated that in the treatment of a tumor inaccordance with the present invention, a formulation containing between0.001 and 5 percent by weight, preferably about 0.01 to 1 percent, willusually constitute a therapeutically effective amount. When administeredsystemically, an amount between 0.01 and 100 mg per kg body weight perday, but preferably about 0.1 to 10 mg per kg, will effect a therapeuticresult in most instances.

In practicing the methods of this invention, the compounds of thisinvention may be used alone or in combination, or in combination withother therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadministered alongwith other compounds typically prescribed for these conditions accordingto generally accepted medical practice. The compounds of this inventioncan be utilized in vivo, ordinarily in mammals, preferably in humans.

In still another embodiment, the anti-inflammatory compounds of theinvention may be coupled to chemical moieties, including proteins thatalter the functions or regulation of target proteins for therapeuticbenefit. These proteins may include in combination other inhibitors ofcytokines and growth factors that may offer additional therapeuticbenefit in the treatment of inflammatory disorders. In addition, theanti-inflammatory compounds of the invention may also be conjugatedthrough phosphorylation to biotinylate, thioate, acetylate, iodinateusing any of the cross-linking reagents well known in the art.

F. Molecular Biology, Microbiology and Recombinant DNA Techniques

In accordance with the present invention, as described above or asdiscussed in the Examples below, there may be employed conventionalmolecular biology, microbiology and recombinant DNA techniques. Suchtechniques are explained fully in the literature. See for example,Sambrook et al., (1989) Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Press; Glover, (1985) DNA Cloning: A Practical Approach;Gait, (1984) Oligonucleotide Synthesis; Harlow & Lane, (1988)Antibodies—A Laboratory Manual, Cold Spring Harbor Press; Roe et al.,(1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley;and Ausubel et. al., (1995) Current Protocols in Molecular Biology,Greene Publishing.

G. Antisense RNA

Antisense molecules are RNA or single-stranded DNA molecules withnucleotide sequences complementary to a specified mRNA. When alaboratory-prepared antisense molecule is injected into cells containingthe normal mRNA transcribed by a gene under study, the antisensemolecule can base-pair with the mRNA, preventing translation of the mRNAinto protein. The resulting double-stranded RNA or RNA/DNA is digestedby enzymes that specifically attach to such molecules. Therefore, adepletion of the mRNA occurs, blocking the translation of the geneproduct so that antisense molecules find uses in medicine to block theproduction of deleterious proteins. Methods of producing and utilizingantisense RNA are well known to those of ordinary skill in the art (see,for example, Lichtenstein & Nellen (Editors), Antisense Technology: APractical Approach, Oxford University Press, 1997; Agrawal & Crooke,Antisense Research and Application (Handbook of ExperimentalPharmacology, Vol. 131), Springer Verlag, 1998; Gibson, Antisense andRibozyme Methodology: Laboratory Companion, Chapman & Hall, 1997; Mol &Van Der Krol, Antisense Nucleic Acids and Proteins, Marcel Dekker;Weiss, Antisense Oligodeoxynucleotides and Antisense RNA: NovelPharmacological and Therapeutic Agents, CRC Press, 1997; Stanley et al.,(1993) Antisense Research and Applications, CRC Press; Stein & Krieg,(1998) Applied Antisense Oligonucleotide Technology).

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize antisense RNAconstitutively or inducibly can be introduced into cell lines, cells, ortissues. RNA molecules may be modified to increase intracellularstability and half-life. Possible modifications include, but are notlimited to, the addition of flanking sequences at the 5′ and/or 3′ endsof the molecule or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept can be extended by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

H. Fusion Proteins

A fusion protein is an expression product resulting from the fusion oftwo genes. Such a protein may be produced, e.g., in recombinant DNAexpression studies or, naturally, in certain viral oncogenes in whichthe oncogene is fused to gag.

The production of a fusion protein sometimes results from the need toplace a cloned eukaryotic gene under the control of a bacterial promoterfor expression in a bacterial system. Sequences of the bacterial systemare then frequently expressed linked to the eukaryotic protein. Fusionproteins are used for the analysis of structure, purification, function,and expression of heterologous gene products.

A fused protein is a hybrid protein molecule which can be produced whena nucleic acid of interest is inserted by recombinant DNA techniquesinto a recipient plasmid and displaces the stop codon for a plasmidgene. The fused protein begins at the amino end with a portion of theplasmid protein sequence and ends with the protein of interest.

The production of fusion proteins is well known to one skilled in theart (See, e.g., U.S. Pat. Nos. 5,908,756; 5,907,085; 5,906,819;5,905,146; 5,895,813; 5,891,643; 5,891,628; 5,891,432; 5,889,169;5,889,150; 5,888,981; 5,888,773; 5,886,150; 5,886,149; 5,885,833;5,885,803; 5,885,779; 5,885,580; 5,883,124; 5,882,941; 5,882,894;5,882,864; 5,879,917; 5,879,893; 5,876,972; 5,874,304; and 5,874,290).For a general review of the construction, properties, applications andproblems associated with specific types of fusion molecules used inclinical and research medicine, see, e.g., Chamow et al., (1999)Antibody Fusion Proteins, John Wiley.

I. Peptide Mimetics

This invention also includes peptide mimetics, e.g., peptide mimeticswhich mimic the three-dimensional structure of the NBD on IKKβ and blockNEMO binding at the NBD by binding to NEMO. Such peptide mimetics mayhave significant advantages over naturally-occurring peptides,including, for example, more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, and efficacy), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others.

In one form, mimetics are peptide-containing molecules that mimicelements of protein secondary structure. See, for example, Johnson etal., (1993) Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzutoet al., (Editors) Chapman & Hall. The underlying rationale behind theuse of peptide mimetics is that the peptide backbone of proteins existschiefly to orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. In another form, peptide analogs are commonly used inthe pharmaceutical industry as non-peptide drugs with propertiesanalogous to those of the template peptide. These types of non-peptidecompounds are also referred to as “peptide mimetics” or“peptidomimetics” (Fauchere, (1986) Adv. Drug Res. 15, 29-69; Veber &Freidinger, (1985) Trends Neurosci. 8, 392-396; and Evans et al., (1987)J. Med. Chem. 30, 1229-1239, which are incorporated herein by reference)and are usually developed with the aid of computerized molecularmodeling.

Peptide mimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptide mimetics are structurallysimilar to a paradigm polypeptide (i.e., a polypeptide that has abiochemical property or pharmacological activity), such as the NBD, buthave one or more peptide linkages optionally replaced by a linkageselected from the group consisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methodsknown in the art and further described in the following references:Weinstein, (1983) Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Marcel Dekker; Morley, (1980) Trends Pharmacol. Sci. 1,463-468 (general review); Hudson et al., (1979) Int. J. Pept. ProteinRes. 14, 177-185 (—CH₂NH—, CH₂CH₂—); Spatola et al., (1986) Life Sci.38, 1243-1249 (—CH₂—S); Hann, (1982) J. Chem. Soc. Perkin Trans. 1,307-314 (—CH—CH—, cis and trans); Almquist et al., (1980) J. Med. Chem.23, 1392-1398 (—COCH₂—); Jennings-White et al., (1982) Tetrahedron Lett.23, 2533 (—COCH₂—); U.S. Pat. No. 4,424,207 (—CH(OH)CH₂—); Holladay etal., (1983) Tetrahedron Lett. 24, 4401-4404 (—C(OH)CH₂—); and Hruby,(1982) Life Sci. 31, 189-199 (—CH₂—S—); each of which is incorporatedherein by reference.

Labeling of peptide mimetics usually involves covalent attachment of oneor more labels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptide mimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., are not contact pointsin NBD-NEMO complexes) to which the peptide mimetic binds to produce thetherapeutic effect. Derivitization (e.g., labeling) of peptide mimeticsshould not substantially interfere with the desired biological orpharmacological activity of the peptide mimetic. NBD peptide mimeticscan be constructed by structure-based drug design through replacement ofamino acids by organic moieties (see, for example, Hughes, (1980)Philos. Trans. R. Soc. Lond. 290, 387-394; Hodgson, (1991) Biotechnol.9, 19-21; Suckling, (1991) Sci. Prog. 75, 323-359).

The use of peptide mimetics can be enhanced through the use ofcombinatorial chemistry to create drug libraries. The design of peptidemimetics can be aided by identifying amino acid mutations that increaseor decrease binding of a NBD (e.g., the NBD on IKKβ) to NEMO. Forexample, such mutations as identified in Table 1. Approaches that can beused include the yeast two hybrid method (see Chien et al., (1991) Proc.Natl. Acad. Sci. USA 88, 9578-9582) and using the phage display method.The two hybrid method detects protein-protein interactions in yeast(Fields et al., (1989) Nature 340, 245-246). The phage display methoddetects the interaction between an immobilized protein and a proteinthat is expressed on the surface of phages such as lambda and M13(Amberg et al., (1993) Strategies 6, 2-4; Hogrefe et al., (1993) Gene128, 119-126). These methods allow positive and negative selection forprotein-protein interactions and the identification of the sequencesthat determine these interactions.

For general information on peptide synthesis and peptide mimetics, see,for example, Jones, (1992) Amino Acid and Peptide Synthesis, OxfordUniversity Press; Jung, (1997) Combinatorial Peptide and NonpeptideLibraries: A Handbook, John Wiley; and Bodanszky et al., (1993) PeptideChemistry: A Practical Textbook, 2nd Revised Edition, Springer Verlageach of which is hereby incorporated in its entirety.

J. Transgenic Animals

Transgenic animals are genetically modified animals into whichrecombinant, exogenous or cloned genetic material has beenexperimentally transferred. Such genetic material is often referred toas a transgene. The nucleic acid sequence of the transgene may beintegrated either at a locus of a genome where that particular nucleicacid sequence is not otherwise normally found or at the normal locus forthe transgene. The transgene may consist of nucleic acid sequencesderived from the genome of the same species or of a different speciesthan the species of the target animal.

The term “germ cell line transgenic animal” refers to a transgenicanimal in which the genetic alteration or genetic information wasintroduced into a germ line cell, thereby conferring the ability of thetransgenic animal to transfer the genetic information to offspring. Ifsuch offspring in fact possess some or all of that alteration or geneticinformation, then they too are transgenic animals.

The alteration or genetic information may be foreign to the species ofanimal to which the recipient belongs, foreign only to the particularindividual recipient, or may be genetic information already possessed bythe recipient. In the last case, the altered or introduced gene may beexpressed differently than the native gene.

Transgenic animals can be produced by a variety of different methodsincluding transfection, electroporation, microinjection, gene targetingin embryonic stem cells and recombinant viral and retroviral infection(see, e.g., U.S. Pat. No. 4,736,866; U.S. Pat. No. 5,602,307; Mullins etal., (1993) Hypertension 22, 630-633; Brenin et al., (1997) Surg. Oncol.6, 99-110; Tuan, (1997) Recombinant Gene Expression Protocols, Methodsin Molecular Biology No. 62, Humana Press).

A number of recombinant or transgenic mice have been produced, includingthose which express an activated oncogene sequence (U.S. Pat. No.4,736,866); express simian SV40 T-antigen (U.S. Pat. No. 5,728,915);lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Pat.No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Pat. No.5,723,719); express at least one human gene which participates in bloodpressure control (U.S. Pat. No. 5,731,489); display greater similarityto the conditions existing in naturally occurring Alzheimer's disease(U.S. Pat. No. 5,720,936); have a reduced capacity to mediate cellularadhesion (U.S. Pat. No. 5,602,307); possess a bovine growth hormone gene(Clutter et al., (1996) Genetics 143, 1753-1760) or, are capable ofgenerating a fully human antibody response (Zou et al., (1993) Science262, 1271-1274).

While mice and rats remain the animals of choice for most transgenicexperimentation, in some instances it is preferable or even necessary touse alternative animal species. Transgenic procedures have beensuccessfully utilized in a variety of non-murine animals, includingsheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits,cows and guinea pigs (see Kim et al., (1997) Mol. Reprod. Dev. 46,515-526; Houdebine, (1995) Reprod. Nutr. Dev. 35, 609-617; Petters,(1994) Reprod. Fertil. Dev. 6, 643-645; Schnieke et al., (1997) Science278, 2130-2133; Amoah, (1997) J. Animal Science 75, 578-585).

The method of introduction of nucleic acid fragments into recombinationcompetent mammalian cells can be by any method which favorsco-transformation of multiple nucleic acid molecules. Detailedprocedures for producing transgenic animals are readily available to oneskilled in the art, including the disclosures in U.S. Pat. No. 5,489,743and U.S. Pat. No. 5,602,307.

The present invention comprises transgenic animals expressing a geneencoding a NBD, and mutations of that gene resulting in conservative andnon-conservative amino acid substitutions when compared to the wild typegene.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, are herebyincorporated by reference.

EXAMPLES Example 1 Identification of NEMO Binding Domain on IKKB

To identify the NEMO-interacting domain of IKKβ we performed in vitropull down assays (Zhong et al., (1997) Cell 89, 413-424) using abacterially expressed version of full length NEMO fused at itsNH2-terminus to glutathione S-transferase (GST-NEMO; FIG. 1A). Varioustruncation mutants lacking different functional domains of IKKβ(catalytic domain, leucine zipper and helix-loop-helix; FIG. 1A) wereconstructed.

All sub-cloning and mutagenesis of full length cDNA clones of IKKα andIKKβwas performed by PCR using cloned Pfu DNA-polymerase (Stratagene).The wild-type and mutated IKKβ cDNA were inserted into the KpnI and NotIrestriction sites of pcDNA-3 or pcDNA-3.1-xpress (Invitrogen) and allIKKα cDNAs were inserted into the EcoRI and XhoI sites of the samevectors. FLAG-tagged versions of both kinases were constructed bysubcloning into pFLAG-CMV-2 (Sigma). For GST-IKKβ-(644-756), the PCRfragment was inserted into the EcoRI and XhoI sites of pGEX-4T1(Pharmacia). Full length cDNA encoding human NEMO was obtained byreverse transcriptase (RT)-PCR from HeLa cell mRNA using the Expand™Long Template PCR System (Boehringer Mannheim) and the primer pair(5′-ATAGACGAATTCAATAGGCACCTCTGGAAG) (SEQ ID NO: 20) and(3′-TAGGACCTCGAGCTACTCAATGCACTCCATG) (SEQ ID NO: 21). The resulting PCRfragment was inserted into the EcoRI and XhoI sites of pcDNA-3 orpcDNA-s.1-xpress. All subsequent NEMO mutants were constructed by PCRusing Pfu DNA-polymerase. GST-NEMO was constructed by sub-cloning thefull-length cDNA into the EcoRI and XhoI sites of pGEX-4T1.

These mutants were labeled by in vitro translation with [³⁵S]-methionine(input; FIG. 1B), mixed with either GST alone or GST-NEMO, andprecipitated using glutathione-agarose. None of the mutants interactedwith GST alone, whereas wild-type and all three NH2-terminal truncationsof IKKβ (307-756, 458-756 and 486-756) interacted with GST-NEMO (FIG. 1B(right panel). In contrast, none of the COOH-terminal truncation mutants(1-456, 1-605 or 1-644) precipitated with GST-NEMO. These resultsdemonstrate that NEMO interacts with a region in the COOH-terminus ofIKKβdistal to the helix-loop-helix (HLH) domain. A mutant consisting ofonly the region from amino acid 644 (immediately after the HLH) to theCOOH-terminus (residue 756) of IKKβ was constructed next. As shown inFIG. 1C, this mutant did not precipitate with GST but did interact withGST-NEMO confirming that this region mediates the interaction betweenthese two molecules.

The effects of IKKβ-(644-756) on IL-1β- and TNFα-induced NF-κBactivation by transiently transfecting HeLa cells with the mutanttogether with an NF-κB-dependent reporter plasmid (pBIIX-luciferase) wastested next (Kopp & Ghosh, (1994) Science 265, 956-959). Fortransfection studies, HeLa and COS cells were seeded into eithertwenty-four well (1×10⁵ cells/well) or six well (5×10⁵ cells/well)plates and grown for twenty-four hours before transfection of DNA withFugene6 (Roche) according to the manufacturer's protocol. Cells intwenty-four well and six well trays received a total of 1 μg or 2 μg ofDNA respectively. After forty-eight hours cells were lysed with TNT (200mM NaCl, 20 mM Tris-pH 8.0, 1% Triton-100) and the lysate were used foreither immunoprecipitation or luciferase assay (Primage Luciferase AssaySystem).

FIG. 1D shows that IKKβ-(644-756) inhibited NF-κB activation induced bythese cytokines in a dose-dependent manner. These results indicate thatIKKβ-(644-756) acts as a dominant-negative by titrating endogenous NEMOout of the core IκB-kinase complex. Without the recruitment ofregulatory proteins by NEMO, IKKβ becomes refractory to IL-1β- andTNFα-induced signals that should otherwise cause its activation.Structurally, NEMO consists of extensive α-helical regions containingtwo prominent stretches of coiled-coil and a leucine-zipper motif, and aCOOH-terminal zinc-finger domain (FIG. 2A) (Mercurio et al., (1999) Mol.Cell. Biol. 19, 1526-1538; Yamaoka et al., (1998) Cell 93, 1231-1240;Rothwarf et al., (1998) Nature 395, 297-300). Previous studiesattempting to identify the region of NEMO required for its interactionwith IKKβ have generated conflicting results (Harhaj et al., (1999) J.Biol. Chem. 274, 15297-15300). To address this question GST-pull-downassays using a GST-fusion protein of IKKβ-(644-756) (FIG. 2A) andvarious [³⁵S]-methionine-labeled truncation mutants of NEMO (FIG. 2A)were performed. FIG. 2A (right panel) summarizes the results of theseexperiments in which it was demonstrated that IKKβ-(644-756) interactedwith NEMO-(1-196), -(1-302) and -(44-419) but not NEMO-(197-419) or-(86-419). Identical results were obtained from immuno-precipitationstudies using lysate of COS or HEK293 cells transiently transfected withFLAG-tagged IKKβ and the NEMO mutants (data not shown).

For all immunoprecipitations HeLa or COS cells grown in six well trayswere lysed in 500 μl TNT. FLAG-tagged proteins were precipitated fromlysate of transfected cells for two hours at 4° C. using 20 μl ofanti-FLAG (M2)-conjugated agarose beads (Sigma). Immunoprecipitations ofendogenous IKKβ or NEMO were performed using 1 μg of specific rabbitpolyclonal antibodies (Santa Cruz) plus 20 μl of Protein-A sepharose(Amersham-Pharmacia). For immunoblotting, precipitates were washed threetimes with TNT, twice with PBS then suspended in SDS-sample buffer.Proteins were separated by SDS-PAGE (10%), transferred to PVDF membranesand visualized by enhanced chemiluminesence (Amersham-Pharmacia).

These results establish that the interaction domain lies betweenresidues 44 and 86, a region including the first α-helix of NEMO. Amutant was therefore made in which α-helix up to the first coiled-coildomain was deleted (residues T50-L93; del.αH). This mutant did notinteract with IKKβ-(644-756) (FIG. 2B). Furthermore transfection studiesusing pBIIX-luciferase demonstrated that del.αH inhibited TNFα-inducedNF-κB activity (FIG. 2C) confirming previous reports that theCOOH-terminus of NEMO which can not interact with IKKβ, is adominant-negative inhibitor of NF-κB (Mercurio et al., (1999) Mol. Cell.Biol. 19, 1526-1538; Rothwarf et al., (1998) Nature 395, 297-300). Takentogether, FIGS. 1 and 2 show that the interaction between IKKβ and NEMOoccurs via the COOH-terminus of IKKβ and the first α-helical region ofNEMO. These findings suggest a model in which the NH2-terminus of NEMOanchors it to the IKK-complex leaving the remainder of the moleculecontaining several protein:protein interaction domains free andaccessible for interacting with upstream regulators of IKK function.

Example 2 NEMO Regulation of IKKB Function Through Interaction at NBD

To fully characterize the NEMO-interaction domain of IKKβ furthertruncation mutants between residues V644 and S756 (FIG. 3A) wereconstructed. Immediately after the HLH, the amino acid sequence to thecysteine at position 662 exhibits 72% identity with IKKα (denoted α₁ inFIG. 3A). Following this, the region up to E707 is a serine-rich domainpreviously reported to be a target for auto-phosphorylation and tofunction in down-regulating IKKβ activity after stimulation bypro-inflammatory cytokines (Delhase et al., (1999) Science 284,309-313). The sequence succeeding this contains no serine residues untilposition 733. Mutants sequentially omitting each of these regions were[³⁵S]-methionine-labeled and used in GST-pull-down assays as describedabove. FIG. 3A summarizes the results from these experimentsdemonstrating that none of the IKKβ mutants precipitated with GST-NEMOand indicating that the interaction domain resides in the extremeCOOH-terminus between residues F734 and S756.

Comparison of this short segment of IKKβ with the corresponding regionof IKKα reveals two striking structural characteristics (FIG. 3B).First, the sequence from F734 to T744 of IKKβ (α2 in FIG. 3B) isidentical to the equivalent sequence in IKKα (L737 to L742 of IKKβ andL738 to L743 of IKKα). Second, the sequence of IKKβ extends beyond theCOOH-terminal residue of IKKα (E745) for twelve amino acids comprising ahighly acidic region in which five of the residues are glutamates (FIG.3B). The marked similarity between the α2-region of IKKβ and the extremeCOOH-terminus of IKKα together with previous reports that NEMO does notinteract with IKKα in vitro (Mercurio et al., (1999) Mol. Cell. Biol.19, 1526-1538; Yamaoka et al., (1998) Cell 93, 1231-1240; Rothwarf etal., (1998) Nature 395, 297-300) led to the hypothesis that theNEMO-interaction domain would be the glutamate-rich portion of IKKβ(E745 to S756).

To test this hypothesis, a truncation mutant omitting this region wasmade (1-744; FIG. 3C) and investigated for its ability to interact withGST-NEMO. The mutant associated with GST-NEMO to an equal extent aswild-type IKKβ (FIG. 3C); these results have been confirmed byco-immunonoprecipitating epitope-tagged NEMO and IKKβ-(1-744) fromlysate of transiently transfected COS cells. These findings demonstratethat the NEMO-interaction domain of IKKβ is within the α2-region of theCOOH-terminus.

IKKβ COOH-terminal truncation mutants were next used to test the effectsof NEMO association on basal and induced activity of IKKβ. FIG. 3D showsthat truncation of IKKβ at V644, eliminating the serine-rich region (seeFIG. 3A), resulted in complete loss of basal auto-phosphorylation. Incontrast, a mutant containing the serine-rich region (1-733), exhibitedsignificantly higher levels of auto-phosphorylation than wild-type IKKβ(FIG. 3D). Intriguingly, the level of auto-phosphorylation ofIKKβ-(1-744) which contains the NEMO-binding α2-region, was identical tothat observed with the wild-type kinase. To test the effects that thesemutations have on basal kinase activity, mutants were transientlytransfected into HeLa cells and NF-κB activity determined by luciferaseassay as described in Example 1. The results in FIG. 3D demonstrate thatIKKβ-(1-644) did not induce NF-κB activity whereas IKKβ-(1-733) causedincreased activation compared with wild-type (FIG. 3E). Furthermore,NF-κB activity induced by IKKβ-(1-744) was identical to that induced bywild-type IKKβ.

These results demonstrate that basal auto-phosphorylation and kinaseactivity of IKKβ is dependent on the ability of NEMO to associate withthe kinase. One explanation for these observations may be that NEMOrecruits a phosphatase to the IKK-complex that regulates basal IKKβfunction by targeting the serine-rich region of the COOH-terminus.Inability to bind NEMO therefore prevents phosphatase recruitment andcauses increased phosphorylation within this region.

To directly test the effect that loss of the α2-region has on thecatalytic activity of IKKβ, an immune-complex kinase assay was performedon lysate from transfected HeLa cells (FIG. 3F). For immune-complexkinases assays, precipitates were washed with TNT then with kinasebuffer (20 mM HEPES pH 7.5, 20 mM MgCl2, 1 mM EDTA, 2 mM NaF, 2 mMβ-glycerophosphate, 1 mM DTT, 10 μM ATP). Precipitates were thenincubated for fifteen minutes at 30° C. in 20 μl of kinase buffercontaining GST-IκBα-(1-90) and 10 μCi [³²P]-γ-labeled ATP(Amersham-Pharmacia). The substrate was precipitated usingglutathione-agarose (Amersham-Pharmacia) and separated by SDS-PAGE(10%). Kinase activity was determined by autoradiography. Phosphorylatedproteins associated with the kinase complex appeared on autoradiographsbecause the immuno-precipitated complex was not removed prior toGST-substrate precipitation. Activity of IKKβ (wild-type) was low inuntreated cells but was markedly enhanced after treatment with TNFα.Consistent with the data presented in FIG. 3E, basal activity ofIKKβ-(1-733) was significantly higher than wild-type, however thisactivity was not further enhanced by treatment with TNFα (FIG. 3F).Furthermore, basal and TNFα-induced catalytic activity of IKKβ-(1-744)was identical to the activity of IKKβ (WT). In addition tophosphorylated GST-IκBα, auto-phosphorylated IKKβ proteins were alsodetected (FIG. 3F, top bands). Following TNFα treatment, IKKβ (WT) andIKKβ-(1-744) became rapidly autophosphorylated whereas the already highbasal phosphorylation of IKKβ-(1-733) was only slightly enhanced (FIG.3F). A previous study showed that auto-phosphorylation serves todown-regulate TNFα-induced IKKβ activity by causing conformationalchanges within the protein (Delhase et al., (1999) Science 284,309-313). Taken together, these findings (FIGS. 3D-F) demonstrate thatin the absence of NEMO, IKKβ becomes auto-phoshorylated, basally activeand refractory to TNFα-induced signals indicating that NEMO plays afundamental role in the down-regulation as well as activation of IKKβactivity.

An additional band representing a phosphorylated protein appeared onlyin the samples from TNFα-induced IKKβ (WT) and IKKβ-(1-744) transfectedcells (FIG. 3F). The molecular weight of this protein (49 kDa) stronglysuggests that it is endogenous NEMO associated with the precipitatedcomplex. This is supported by the absence of the band in eitherprecipitate (+/−TNFα) from IKKβ-(1-733) transfected cells. This proteinhas been identified as phosphorylated NEMO by dissociating theprecipitated complex in SDS and re-immunoprecipitating [³²P]-labeledNEMO using specific anti-NEMO antibodies. Induced phosphorylation ofNEMO may therefore represent a further level of regulation of theactivity of the IKK complex.

Example 3 Identification of the NBD on IKKA

Since the α2-region of IKKβ strongly resembles the COOH-terminus of IKKα(FIG. 3B), the ability of IKKα to interact with NEMO was tested.

Immunoprecipitations from lysate of COS cells transiently transfectedwith xpress-tagged NEMO together with FLAG-tagged versions of eitherIKKα or IKKβ were performed using anti-FLAG as described in Example 1.FIG. 4A shows that NEMO interacted equally well with both IKKβ and IKKα.It is possible that in this experiment the interaction with IKKα is notdirect but due instead to the formation of a complex containingendogenous IKKβ, FLAG-IKKα and xpress-NEMO. A GST-pull-down assays wastherefore performed using GST-NEMO and [³⁵S]-methionine-labeled versionsof either wild-type IKKα or a truncated IKKα mutant lacking the eightCOOH-terminal amino acids (1-737: FIG. 4B). In agreement with thefindings presented above (FIG. 4A), but in contrast to previous reports(Mercurio et al., (1999) Mol. Cell. Biol. 19, 1526-1538; Yamaoka et al.,(1998) Cell 93, 1231-1240; Rothwarf et al., (1998) Nature 395, 297-300),wild-type IKKα interacted with NEMO in vitro whereas the truncatedmutant did not (FIG. 4B). These results not only demonstrate that IKKαinteracts with NEMO but also shows that it does so via the COOH-terminalregion containing the six amino acids shared between IKKα and theα2-region of IKKβ (FIG. 3B). Gene-targeting studies have demonstratedprofound differences in the activation of IKKα and IKKβ by TNFα(Woronicz et al., (1997) Science 278, 866-869; Zandi et al., (1997) Cell91, 243-252; Mercurio et al., (1997) Science 278, 860-866; DiDonato etal., (1997) Nature 388, 548-554; Régnier et al., (1997) Cell 90,373-383).

The present findings indicate that the basis of this difference is notdue to differential recruitment of NEMO (Delhase et al., (1999) Science284, 309-313; Takeda et al., (1999) Science 284, 313-316; Hu et al.,(1999) Science 284, 316-20; Li et al., (1999) Science 284, 321-325; Liet al., (1999) J. Exp. Med. 189, 1839-1845; Li et al., (1999) Genes Dev.13, 1322-1328; Tanaka et al., (1999) Immunity 10, 421-429). Instead thedifference most likely lies in the ability of each kinase to integrateNEMO-associated signaling components into an activation response,presumably through differences in the inherent regulatory features ofthe individual kinases.

Further evidence that this short COOH-terminal sequence constitutes theNEMO-interaction domain of the IKKs was obtained when we tested theability of the recently described IKK-related kinase IKKi (Shimada etal., (1999) Int. Immunol. 11, 1357-1362) to interact with NEMO. Sequencecomparison with IKKα and IKKβ (Shimada et al., (1999) Int. Immunol. 11,1357-1362; Woronicz et al., (1997) Science 278, 866-869; Zandi et al.,(1997) Cell 91, 243-52; Mercurio et al., (1997) Science 278, 860-866;DiDonato et al., (1997) Nature 388, 548-554; Régnier et al., (1997) Cell90, 373-383) reveals that IKKi does not contain the α2-region in itsCOOH-terminus (Shimada et al., (1999) Int. Immunol. 11, 1357-1362) andconsistent with this being the NEMO binding domain we found that IKKidoes not interact with GST-NEMO in pull down assays (FIG. 4C). Thisfinding indicates that NEMO is not required for the functional activityof IKKi and this is supported by the inability of IKKi to respond tosignals induced by either TNFα or IL-1β (Shimada et al., (1999) Int.Immunol. 11, 1357-1362).

Example 4 Mutation of Amino Acid Residues in the NBD

Having determined that the α2-region of IKKβ, and the equivalent sixamino acid sequence of IKKα are critical for interaction with NEMO[designated NEMO binding domain (NBD)], a deletion mutant in IKKβlacking the six amino acids from L737 to L742 (del.NBD) was constructed.This deletion mutant did not associate with GST-NEMO (FIG. 4D).Examination of predicted structural and biochemical features of the NBDin context with surrounding residues suggests that it constitutes aninflexible hydrophobic “pocket” within a hydrophilic region of the IKKβCOOH-terminus (FIG. 4E). This suggests a model in which the NBD becomesburied within the first α-helical region of bound NEMO (FIG. 2)preventing its exposure to an aqueous environment thereby maintaining astrong inter-molecular interaction. Whether the interaction is indeed afunction of this hydrophobicity remains to be determined, however wefound that substitution of either W739 or W741 with alanine preventedassociation of NEMO with IKKβ (FIG. 4F). Therefore each of thesehydrophobic tryptophan residues is critical for maintaining a functionalNBD. In addition, mutation of D738 to alanine also prevented NEMOinteraction indicating that a negatively charged residue at thisposition is required for NBD function. In contrast to these mutations,substitution of L737, S740 or L742 with alanine did not affect NEMObinding. To test the effects of these alanine substitutions on IKKβfunction, HeLa cells were co-transfected with each of the point mutantstogether with pBIIX-luciferase reporter. Consistent with the observationthat the basal activity of IKKβ is increased in the absence ofassociated NEMO, IKKβ-(1-733) (FIG. 3E), mutants that did not bind NEMO(D738A, W739A and W741A) activated NF-κB to a greater extent thanwild-type IKKβ or IKKβ-(1-744) (FIG. 4G). In contrast, mutantscontaining substitutions that did not disrupt NEMO association (L737A,S740A and L742A) induced NF-κB to the same level as the controls. Theseresults indicate that NEMO plays a critical role in the down-regulationof intrinsic IKKβ activity.

Further mutations in the NBD were analyzed (see Table 1) for theirability to affect NEMO binding to IKKβ using the GST pulldown assayexplained in Example 3.

TABLE 1 Characterized NBD mutants and their ability to bind to NEMO. NBDBinds to Mutants- NEMO SEQ ID NO: LDWSWL yes  2 LDASAL no  3 ADWSWL yes 4 LDWSWA yes  5 LAWSWL no  7 LEWSWL yes  8 LNWSWL yes  9 LDASWL no 10LDFSWL yes 11 LDYSWL yes 12 LDWSAL no 13 LDWSFL yes 14 LDWSYL no 15LDWAWL yes 16 LDWEWL yes 17 * The substituted amino acid residue isindicated by bold face.

Example 5 Agents which Interact with NBD to Block NEMO Binding

The relatively small size of the NBD makes it an attractive target forthe development of compounds aimed at disrupting the core IKK complex.The relevance of this approach was investigated by designingcell-permeable peptides spanning the IKKβNBD and determining theirability to dissociate the IKKβ-NEMO interaction.

The sequences of the two NBD peptides used in this study were[DRQIKIWFQNRRMKWKK]TALDWSWLQTE (wild-type) (SEQ ID NO: 18) and[DRQIKIWFQNRRMKWKK]TALDASALQTE (mutant) (SEQ ID NO: 19). Theantennapedia homeodomain sequence (Derossi et al., (1994) J. Biol. Chem.269, 10444-10450; U.S. Pat. No. 5,888,762; U.S. Pat. No. 6,015,787; U.S.Pat. No. 6,080,724) is bracketed and the positions of the Wto Amutations are underlined. Both peptides were dissolved in DMSO to astock concentration of 20 mM. For all experiments DMSO alone controlswere no different from no peptide controls.

The wild-type NBD peptide consisted of the region from T735 to E745 ofIKKβfused with a sequence derived from the third helix of theantennapedia homeodomain that has been shown to mediate membranetranslocation (Derossi et al., (1994) J. Biol. Chem. 269, 10444-10450).The mutant was identical except that the tryptophan residues (W739 andW741) in the NBD were mutated to alanine. FIG. 5A shows that the NBD(WT) but not the mutant dose-dependently inhibited in vitro pull-down of[³⁵S]-labeled IKKβ by GST-NEMO and [³⁵S]-labeled NEMO byGST-IKKβ-(644-756). To test the ability of the NBD peptides to entercells and inhibit the IKKβ-NEMO interaction, HeLa cells were incubatedwith the peptides for different time periods and immunoprecipitated theIKK complex using anti-NEMO. In agreement with the in vitro data (FIG.5A), wild-type but not mutant disrupted the formation of the endogenousIKK complex (FIG. 5B).

Example 6 Agents which Block NEMO Function

The effects of the NBD peptides on signal-induced activation of NF-κBwere investigated next. After transfecting HeLa cells with thepBIIX-luciferase reporter, cells were preincubated with wild-type ormutant peptides, treated with TNFα and NF-κB activation measured by theluciferase reporter assay. As shown in FIG. 5C (left panel), thewild-type NBD peptide inhibited TNFα-induced NF-κB activation whereasthe mutant had no effect. Interestingly, the basal NF-κB activity wasenhanced by treatment with the wild-type peptide (FIG. 5C; right panel),a finding which concurs with results from previous mutational analysis(FIGS. 3E-F and 4G). This indicates that removal of NEMO increases thebasal, intrinsic activity of IKK, while abolishing its responsiveness toTNFα. Further analysis using electrophoretic mobility shift assays(EMSA) also demonstrated that only the wild-type NBD peptide inhibitedTNFα-induced activation and nuclear translocation of NF-κB (FIG. 5D).Taken together these results demonstrate that delivery of an intact NBDpeptide into cells disrupts the IKKβ-NEMO interaction and preventspro-inflammatory signals from activating NF-κB. In contrast,transduction with a peptide containing mutations at the tryptophanresidues that are critical for maintaining the NEMO interaction has noeffect.

Example 7 Agents Capable of Down-Regulating E-Selectin

Many proteins involved in the initiation and maintenance of inflammatoryresponses require NF-κB activation for induced expression of their genes(Ghosh et al., (1998) Annu. Rev. Immunol. 16, 225-260; May & Ghosh,(1998) Immunol. Today 19, 80-88). One such protein, E-selectin, is aleukocyte adhesion molecule expressed on the luminal surface of vascularendothelial cells after activation by pro-inflammatory stimuli such asIL-1 or TNFα (Pober et al., (1986) J. Immunol. 436, 1680-1687;Bevilacqua et al., (1987) Proc. Natl. Acad. Sci. USA 84, 9238-9242;Collins et al., (1995) FASEB J. 9, 899-909). Expression of E-selectinand other NF-κB-dependent adhesion molecules is crucial for the arrestand recruitment of leukocytes into sites of acute and chronicinflammation. To assess the anti-inflammatory potential of the NBDpeptide, primary human umbilical vein endothelial cells (HUVEC) werepretreated with the wild-type and mutant peptides and E-selectinexpression induced with TNFα. Consistent with the effects on basal NF-κBactivation (FIG. 5C), the wild-type NBD peptide induced low levelexpression of E-selectin (FIG. 5E). However, after TNFα-treatment thewild-type but not mutant significantly reduced expression of E-selectin(FIG. 5E). Inhibition by wild-type NBD peptide reduced expression to thelevel induced by the peptide in the absence of TNFα.

The importance of the present invention can be viewed on two levels.First, Applicants have identified the structural requirements for theassociation of NEMO with the IKKs and found that association with IKKβis dependent on three amino acids (D738, W739 and W741) within the NBD.Furthermore, NEMO not only functions in the activation of IKKβ but italso has a critical role in suppressing the intrinsic, basal activity ofthe IKK complex. The second level of importance is the obvious clinicaluse for drugs targeting the NBD. Applicants have demonstrated that acell-permeable peptide encompassing the NBD is able to not only inhibitTNFα-induced NF-κB activation but also reduce expression of E-selectin,an NF-κB-dependent target gene, in primary human endothelial cells. TheNBD is only six amino acids long, and therefore it is within the abilityof one skilled in the art to design peptido-mimetic compounds thatdisrupt the core IKK complex. Since the effect of disrupting the complexis to increase the basal activity of the IKK, treatment with anNBD-targeting compound can avoid issues of toxicity, e.g., due tohepatocyte apoptosis, that might arise from administering drugs thatcompletely abolish the activity of NF-κB. Hence, identification of theNBD is a means for the development of novel anti-inflammatory drugs thatprevent activating signals from reaching the IKK complex, yet maintain alow level of NF-κB activity and avoid potential toxic side-effects.

Example 8 NBD Peptide-Mediated Inhibition of Inflammatory Response InVivo

The NBD peptide was tested for its ability to inhibit inflammatoryresponses in animals using two distinct models of acute inflammation. Inthe first model, ear edema was induced in mice usingphorbol-12-myristate-13-acetate (PMA) and the effects of topicaladministration of the NBD peptides were measured. Ear edema using PMAwas induced in replicate groups of age and sex matched mice aspreviously described (Chang et al., (1987) Eur. J. Pharmacol. 142,197-205). Twenty μl of either NBD peptides (200 μg/ear), dexamethasone(40 μg/ear) or vehicle (DMSO:Ethanol; 25:75 v/v) was applied topicallyto the right ear of mice thirty minutes before and thirty minutes afterthe application of 20 μl of PMA (5 μg/ear) dissolved in ethanol. Earswelling was measured six hours after PMA application using a microgaugeand expressed as the mean difference in thickness between the treated(right) and untreated (left) ears. Statistical analysis of the data wasperformed using the students t-test. A value of p<0.05 was consideredstatistically significant.

FIG. 6A shows that the wild type peptide significantly reduced (77±3%inhibition; p<0.05) PMA-induced ear thickening to the level observedwith dexamethosone (82±9% inhibition; p<0.05). In contrast, the effectobserved with an equivalent dose of mutant was insignificant (p=0.09).Neither peptide had an effect when administered in the absence of PMA(not shown).

In a second model, peritonitis was induced in mice by intraperitoneal(i.p.) injection of zymosan either alone or in combination withdexamethasone or the NBD peptides. For zymosan-induced peritonitis,measurement of peritoneal exudates and inflammatory cell collectionsfrom replicate groups of age and sex matched mice (C57BL/6NCR) wereperformed as previously described (Getting et al., (1998) Immunology 95,625-630). Groups of animals were injected concomitantly with one mlzymosan (1 mg/ml) and either dexamethasone (100 mg/ml) or the NBDpeptides (200 mg/ml). The concentration of NOX (nitrate plus nitrite)present in the inflammatory exudates was measured using a colorimetricassay kit (Alexis Corporation) according to the manufacturers protocol.

As shown in FIG. 6B zymosan injection caused an accumulation ofinflammatory exudate fluids and migration of polymorphonuclear cells(PMN) into the peritoneum of these animals. Treatment of mice with wildtype NBD peptide or dexamethasone significantly reduced exudateformation and PMN accumulation whereas the mutant had no effect.

Various in vivo studies have demonstrated a role for NO in exudateformation and leukocyte migration into inflammatory sites (Ialenti etal., (1992) Eur. J. Pharmacol. 211, 177-182; Ialenti et al., (1993) Br.J. Pharmacol. 110, 701-706; Iuvone et al., (1998) Br. J. Pharmacol. 123,1325-1330). Therefore the effects of the NBD peptides on NOXaccumulation in the peritoneal exudates of zymosan-treated mice wasinvestigated. FIG. 6C (lower panel) shows that dexamethasone andwild-type peptide reduced NOX by 86±7% and 66±4% respectively whereasthe mutant had no effect. These results are consistent with previousstudies demonstrating that reduction of exudate formation and cellaccumulation closely correlate with inhibition of NF-κB activation andreduction of NO formation (D'Acquisto et al., (1999) Eur. J. Pharmacol.369, 223-236; D'Acquisto et al., (1999) Naunyn-Schmeideberg's Arch.Pharmacol. 360, 670-675). Therefore the wild-type NBD peptide is aneffective inhibitor of inflammation in experimental animal models.

Example 9 Inhibition of Osteoclast Differentiation by the NBD Peptide

The processes of bone morphogenesis and remodeling require themaintenance of a balance between the synthesis of bone matrix byosteoblasts and the resorbtion of bone by osteoclasts (Suda et al.,(1992) Endocr. Rev. 13, 66-80; Suda et al., (1999) Endocr. Rev. 20,345-357). Bone-resorbing osteoclasts are multinucleated giant cells thatdifferentiate from myeloid precursors and various soluble factorsincluding colony stimulating factor-1 (CSF-1), Interleukin-1 (IL-1),Tumor necrosis factor-α (TNF-α), IL-6 and IL-11 (Suda et al., (1992)Endocr. Rev. 13, 66-80; Suda et al., (1999) Endocr. Rev. 20, 345-357)that affect osteoclast differentiation at distinct stages. One factorthat is critical for osteoclastogenesis is the recently describedmolecule named RANKL (receptor activator of NF-κB ligand) that is alsoknown as ODF (osteoclast differentiation factor), OPGL (osteoprotegerinligand) and TRANCE (TNF-related activation-induced cytokine) (Kong etal., (1999) Nature, 397, 315-323; Lacey et al., (1998) Cell 93, 165-176;Suda et al., (1999) Endocr. Rev. 20, 345-357; Wong et al., (1999) J.Leukoc. Biol. 65, 715-724; Yasuda et al., (1998) Proc. Natl. Acad. Sci.USA 95, 3597-3602). The receptor for RANKL is a member of theTNF-receptor family named RANK (receptor activator of NF-κB) (Andersonet al., (1997) Nature 390, 175-179; Dougall et al., (1999) Genes Dev.13, 2412-2424) and binding of RANKL induces NF-κB activation (Andersonet al., (1997) Nature 390, 175-179; Darnay et al., (1998) J. Biol. Chem.273, 20551-20555; Darnay et al., (1999) J. Biol. Chem. 274, 7724-31;Suda et al., (1999) Endocr. Rev. 20, 345-357; Wong et al., (1998) J.Biol. Chem. 273, 28355-28359). Moreover, osteoclast differentiation isdependent upon NF-κB activation and gene-targeting studies havedemonstrated that mature osteoclasts fail to develop in mice lacking thep50 and p52 NF-κB subunits (Franzoso et al., (1997) Genes Dev. 11,3482-3496).

Osteoporosis is a severely debilitating disease characterized by anextensive loss of bone mass that is mediated by osteoclast-dependentbone resorbtion (Suda et al., (1992) Endocr. Rev. 13, 66-80; Suda etal., (1999) Endocr. Rev. 20, 345-357). It is therefore possible thatselective inhibition of NF-κB activation in osteoclast precursor cellswould prevent osteoclast differentiation and provide the basis fortherapeutically effective drugs for the treatment of osteoporosis.Therefore the effect of the NBD peptides on osteoclast differentiationwas tested using a previously described in vitro model (Jimi et al.,(1999) Exp. Cell Res. 247, 84-93). Mouse bone marrow cells plated into48-well tissue culture trays were incubated with human macrophage-colonystimulating factor (M-CSF; 20 ng/ml) and human RANKL (100 ng/ml) for sixdays in the absence or presence of various concentrations (6.25, 12.5and 25 mM) of either mutant or wild-type NBD peptide. The cells werethen fixed and stained for the osteoclast phenotypic markertartrate-resistant acid phosphatase (TRAP) and TRAP-positivemultinucleated cells containing more than three nuclei were counted asosteoclasts. Triplicate samples were counted and results were calculatedas means±SD. As shown in FIG. 7 the wild type but not mutant peptidedose-dependently inhibited osteoclast differentiation.

This data demonstrates that disruption of the core IKK complex by a cellpermeable NBD peptide that inhibits NF-κB activation preventsRANKL-induced osteoclast differentiation indicating that drugsspecifically targeting the NBD will be effective for the treatment ofosteoporosis. As an extension of these in vitro studies, the samepeptides can be analyzed for their effects on osteoporosis in vivo.Ovarectomized mice (Charles River Labs) that exhibit severe osteoporosisare treated with the NBD peptides and the effects on bone density over atimecourse of treatment determined.

Example 10 Effect of NBD Peptides on Other NF-κB Mediated Disorders

In addition, it is also possible to examine the effects of the NBDpeptides on asthma. NF-κB activation in bronchiolar epithelial cells,T-cells and bronchiolar macrophages has been observed in the airways ofasthmatic patients and in animal models of asthma (Ray & Cohn, (1999) J.Clin. Invest. 104, 985-993; Christman et al., (2000) Chest 117,1482-1487). In addition, many agents that induce asthma cause NF-κBactivation and many of the genes that encode proteins involved in asthma(i.e., leukocyte adhesion molecules, various chemokines, induciblenitric oxide synthase) are NF-κB-dependent. An established mouse modelof asthma (Kleeberger et al., (1990) Am. J. Physiol. 258, 313-320) canbe used to test the effects of aerosol administration of the NBDpeptides on progression of these conditions associated with asthma. In asimilar manner, the effects of the NBD peptides on septic shock can alsobe measured. Septic shock involves the expression of many NF-κBdependent genes (i.e., TNF, IL-1) that are induced by bacterialendotoxins such as lipopolysaccharide (LPS). LPS comprises the majorconstituents of the cell walls of gram-negative bacteria and is highlyimmunogenic and stimulates the production of endogenous pyrogens IL-1and TNF (Sell et al., (1996) Immunology, Immunopathology & Immunity,Appleton & Lange). To test the effects of the NBD peptides on septicshock, mice are injected with the NBD peptides and LPS and the survivalof animals assessed.

Example 11 Relative Contributions and Importance of Each Amino Acidwithin the NBD to the Interaction with NEMO

As indicated in the foregoing Examples, the NEMO binding domain (NBD) ofIKKα and IKKβ consists of six conserved amino acids (L737 to L742 ofIKKβ and L738 to L743 of IKKα) in the extreme C-terminus of bothkinases. This experiment was performed to obtain a clearer understandingof the relative contributions and importance of each amino acid withinthe foregoing NBDs to the interaction with NEMO. Extensive mutationalanalysis of the IKKβ NBD was performed, in which each residue wassubstituted with various conserved and non-conserved amino acids.

It was determined that substitution of either leucine residue (L737 orL742) or serine 740 did not affect the association of NEMO with IKKβsuggesting that none of these residues play a critical role inmaintaining the interaction. To determine whether multiple mutations ofthese amino acids will affect binding, two mutants were constructed inwhich either L737 and S740 or S740 and L742 (named LS and SLrespectively) were substituted with alanine. GST pull-down and COS celltransfection-immunoprecipitation-immunoblot analysis has revealed thatboth LS and SL mutants associate with NEMO to the same extent as wildtype IKKβ providing further evidence that these residues do notcontribute significantly to the interaction. Furthermore, both LS and SLactivate NF-kB as well as IKKβ when measured by activation of anNF-κB-dependent luciferase reporter construct (pBIIx-luciferase) intransient transfection assays. A double alanine mutant of both leucineresidues (LL) as well as a triple mutant (LSL) may be useful inconfirming the foregoing data regarding the importance of these residuesto NEMO binding.

In contrast to the lack of effects of the mutations described above oneither NEMO binding or NF-κB activation, alanine substitution of theaspartic acid residue within the NBD (D738) prevented IKKβ fromassociating with NEMO. Furthermore, this substitution led to a 2- to3-fold increase in the basal NF-κB-activating ability of IKKβ. Theseresults demonstrate a role for NEMO association in maintaining the basalactivity of the IKK complex. Interestingly, treatment of HeLa cells withthe cell-permeable NBD peptide also led to a modest increase in basalNF-κB activity further supporting the concept that loss of NEMOassociation leads to increased basal IKK activity.

To investigate the nature of the residue at position 738 within the NBD,aspartic acid was substituted with either aspargine (D738N) or glutamicacid (D738E; FIG. 8A). These conservative substitutions maintain eitherthe shape (N) or shape and charge (E) of the residue at this position.As shown in FIG. 8B, neither substitution affected the ability of IKKβto associate with NEMO whereas alanine substitution prevented binding.These data demonstrate that it is the shape (specifically the presenceof second carbon) and not the charge of the side chain of the amino acidat this position that is critical for the interaction between IKKβ andNEMO. Consistent with the previous observations disclosed herein,neither mutation affected the basal activity of IKKβ whereassubstitution with alanine caused an increase in activity (FIG. 8C).

As indicated above, both tryptophan residues within the NBD (W739 andW741) are critical for maintaining the interaction with NEMO. Theeffects of conservative mutations that maintain the aromatic structureof the residues at these positions was investigated by substitutingeither phenylalanine (F) or tyrosine (Y) for tryptophan (FIG. 9A). Inaddition, both tryptophans were mutated to arginine; a non-conservativesubstitution requiring only a single base change within the encodingcodon that is the most common naturally occurring tryptophan mutation.As shown in FIG. 9B, both W739F and W739Y mutants associated with NEMOto the same extent as IKKβ whereas W739R did not bind (FIG. 9C).Together with the effects of alanine substitution (FIG. 9B), thesefindings indicate that the aromatic nature of the residue at thisposition is critical for the function of the NBD. Similar to W739, itwas determined that substitution of W742 with phenylalanine (W742F) didnot affect association with NEMO, whereas mutation to arginine (W742R)prevented binding (FIG. 9D). In contrast to W739, substitution withtyrosine (W742Y) prevented association with NEMO demonstrating that thepresence of a hydroxyl moiety within the amino acid side chain at thisposition is sufficient to prevent association of NEMO. This finding mayindicate the that phosphorylation of a residue within the NBD, albeit anartificially inserted amino acid, prevents association of IKKβ withNEMO.

Example 12 Evaluation of Anti-Inflammatory Compounds in LethalLipopolysaccharide Mouse Model

In this experiment, the ability of anti-inflammatory compounds of theinvention to rescue mice challenged with a lethal amount oflipopolysaccharide (LPS) was assessed. LPS is a bacterial product thatinduces many of the responses that are seen in septic patients,including death. In this model, Salmonella typhimurium LPS inphosphate-buffered serum (PBS) was administered to male C57BL/6 mice byintravenous injection at a dose of 30 mg/kg (600 μg/20 g mouse). Thisdose was established in control experiments to be lethal in 100% of themice that received it. Mice were treated with the test peptide byintravenous injection (in 1% dimethyl sulfoxide in PBS) immediatelyprior to the LPS injection and 24 hours after the LPS injection. Micewere monitored twice daily for up to 8 days after receiving LPS and theduration of survival and the number of surviving mice were recorded.

The results of this study are presented in Table 2 which shows, for eachpeptide and dose, the total number of mice dosed and the number of micesurviving after 5 days.

TABLE 2 compound dose (mg/kg) number dosed survival (day five) 1 5 7 1 25 7 7 8 4 3 5 7 7 8 5 8 8 4 5 8 0 5 1 8 4 5 8 8 8 3 6 1 8 0 5 8 7 8 5 84 8 7 8 5 8 8 8 8 7 5 8 8 8 8 8 8 8 8 8 1 8 0 5 8 7 8 7 8 8 9 1 8 0 5 86 8 8 10 1 8 5 5 8 8 8 8 8 6 11 5 8 6

The results presented in the Table 2 demonstrate that compounds 2, 3,and 5-11 provide significant protection against lethal challenge withLPS in this model when administered at a dose of 5 mg/kg i.v. Compound10 also provided significant protection at a dose of 1 mg/kg i.v.

Example 13 Assessment of Peptides in Concanavalin A-Induced Hepatitis

In this experiment, the ability of anti-inflammatory compounds of theinvention to rescue mice with Concanavalin A-induced hepatitis.

Concanavalin A is a lectin, a class of proteins that bind tocarbohydrates. When carbohydrates are part of a protein, the lectinbinds to the protein. By binding to proteins on the cell surface,concanavalin A stimulates many cells, including T lymphocytes. Inconcert with other mediators that are released by concanavalin Astimulation, these T lymphocytes attack liver cells that also haveconcanavalin A bound to them, causing the liver cells to die. Theinvolvement of T lymphocytes makes this model similar to human viralhepatitis. However, as part of this acute model, there is also a TNFαresponse.

Forty male C57BL/6 mice weighing between 18 g and 22 g were divided intofive treatment groups of eight mice each as shown below.

group treatment 1 Vehicle + vehicle 2 Concanavalin A + vehicle 3Concanavalin A + 0.5 mg/kg compound 10 4 Concanavalin A + 1 mg/kgcompound 10 5 Concanavalin A + 5 mg/kg compound 10

The mice were placed in a restrainer and injected intravenously (i.v.)in the tail with a test peptide or vehicle in 1% DMSO in PBS. The micewere then immediately injected i.v. with 15 mg/kg of concanavalin Adissolved in sterile PBS. The injection volume was 5 ml/kg (100 μI/20 gmouse) with a concanavalin A concentration of 3.0 mg/ml. The nextmorning (18-24 hours later), these mice were euthanized by CO₂inhalation and blood was collected by cardiac puncture. The serum wasthen separated and analyzed for AST and ALT.

The results of this study are shown in Table 3. ALT and AST values aregiven as Sigma-Frankel units/ml, mean±SEM.

TABLE 3 Treatment ALT AST vehicle + vehicle 34 ± 4  121 ± 11concanavalin A + vehicle 1684 ± 21*   938 ± 155 concanavalin A + 0.5mg/kg 1919 ± 234* 1217 ± 192 concanavalin A + 1 mg/kg 1910 ± 281* 1264 ±270 concanavalin A + 5 mg/kg 72 ± 19  99 ± 14

The foregoing results indicate that compound 10 is able to protectagainst concanavalin A-induced liver damage at a dose of 5 mg/kg. Thegross pathology supported this conclusion, suggesting that the liver wasinjured by concanavalin A and that this injury was prevented by compound10 at a dose of 5 mg/kg.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for modulating signal transduction involving IκB in a cell,comprising contacting the cell with an anti-inflammatory compoundcomprising the structure:Xa—Xb, wherein Xa is a membrane translocation domain; Xb is a NF-κBEssential Modulator (NEMO) binding sequence; wherein theanti-inflammatory compound is less than one-hundred amino acids inlength, and wherein the anti-inflammatory compound inhibits NF-κBactivation.
 2. The method of claim 1, wherein the anti-inflammatorycompound further comprises a modifying group.
 3. The method of claim 1,wherein X_(b) comprises the following structure:(Y)_(n)—X_(i)—X₂—X₃—X₄—X₅—X₆-(A)_(m) wherein n and m are each,independently, 0 or 1; A and Y each comprises from 1 to about 3 aminoacid residues; X₁ is Leu, Ala, Ile, or nor-leucine (Nle); X₂ is Asp,Glu, Asn, Gln, homoserine (Hser) or 2-ketopropylalanine (2-ketopropy-A);X₃ is Trp, Phe, Tyr, 4-biphenyl-alanine (Bpa), homophenylalanine (Hphe),2-Naphthylalanine (2-Nal), 1-Naphthylalanine (1-Nal), orcycloxexyl-alanine (Cha); X₄ is Ser, Ala, Glu, Leu, Thr, nor-leucine(Nle), or homoserine (Hser); X₅ is Trp, His, homophenylalanine (Hphe),2-Naphthylalanine (2-Nal), 1-Naphthylalanine (1-Nal), O-benzyl serine(SeroBn), or 3-Pyridylalanine (3-Pal); and X₆ is Leu, Ala, Ile, ornor-leucine (Nle).
 4. The method of claim 3, wherein n is 1 and Y is thesequence Thr-Ala.
 5. The method of claim 3, wherein m is 1 and A is thesequence Gln-Thr-Glu.
 6. The method of claim 1, wherein said compoundcomprises a sequence selected from the group consisting ofThr-Ala-Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:28),Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:29),Thr-Ala-Leu-Asp-Trp-Ser-Trp-Leu (SEQ ID NO:30),Ala-Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:31),Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:32),Leu-Asp-Trp-Ser-Trp-Leu (SEQ ID NO:33),Thr-Ala-Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:34),Thr-Ala-Leu-Asp-Trp-Ser-Trp-Leu-Gln (SEQ ID NO:35),Ala-Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:36),Leu-Asp-Trp-Ser-Trp-Leu-Gln (SEQ ID NO:37),Leu-Asp-Trp-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:38), Ala-Asp-Trp-Ser-Trp-Leu(SEQ ID NO:39), Leu-Asp-Trp-Ser-Trp-Ala (SEQ ID NO:40),Ala-Asp-Trp-Ser-Trp-Ala (SEQ ID NO:41), Leu-Asp-Phe-Ser-Trp-Leu (SEQ IDNO:42), Leu-Asp-Tyr-Ser-Trp-Leu (SEQ ID NO:43), Leu-Asp-Trp-Ala-Trp-Leu(SEQ ID NO:44), Leu-Asp-Trp-Glu-Trp-Leu (SEQ ID NO:45),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:46),Ala-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:47),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Leu (SEQ ID NO:48),Ala-Ala-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:49),Ala-Asp-Trp-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:50),Ala-Asp-Trp-Ser-Trp-Leu (SEQ ID NO:51),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:52),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Leu-Gln (SEQ ID NO:53),Ala-Ala-Asp-Trp-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:54),Ala-Asp-Trp-Ser-Trp-Leu-Gln (SEQ ID NO:55),Ala-Asp-Trp-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:56),Ala-Leu-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:57),Leu-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:58),Thr-Ala-Leu-Asp-Trp-Ser-Trp-Ala (SEQ ID NO:59),Ala-Leu-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:60),Leu-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:61),Leu-Asp-Trp-Ser-Trp-Ala (SEQ ID NO:62),Thr-Ala-Leu-Asp-Trp-Ser-Trp-Ala-Gln-Thr (SEQ ID NO:63),Thr-Ala-Leu-Asp-Trp-Ser-Trp-Ala-Gln (SEQ ID NO:64),Ala-Leu-Asp-Trp-Ser-Trp-Ala-Gln-Thr (SEQ ID NO:65),Leu-Asp-Trp-Ser-Trp-Ala-Gln (SEQ ID NO:66),Leu-Asp-Trp-Ser-Trp-Ala-Gln-Thr (SEQ ID NO:67),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:68),Ala-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:69),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Ala (SEQ ID NO:70),Ala-Ala-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:71),Ala-Asp-Trp-Ser-Trp-Ala-Gln-Thr-Glu (SEQ ID NO:72),Ala-Asp-Trp-Ser-Trp-Ala (SEQ ID NO:73),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Ala-Gln-Thr (SEQ ID NO:74),Thr-Ala-Ala-Asp-Trp-Ser-Trp-Ala-Gln (SEQ ID NO:75),Ala-Ala-Asp-Trp-Ser-Trp-Ala-Gln-Thr (SEQ ID NO:76),Ala-Asp-Trp-Ser-Trp-Ala-Gln (SEQ ID NO:77),Ala-Asp-Trp-Ser-Trp-Ala-Gln-Thr (SEQ ID NO:78),Thr-Ala-Leu-Asp-Phe-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:79),Leu-Asp-Phe-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:80),Thr-Ala-Leu-Asp-Phe-Ser-Trp-Leu (SEQ ID NO:81),Ala-Leu-Asp-Phe-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:82),Leu-Asp-Phe-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:83),Leu-Asp-Phe-Ser-Trp-Leu (SEQ ID NO:84),Thr-Ala-Leu-Asp-Phe-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:85),Thr-Ala-Leu-Asp-Phe-Ser-Trp-Leu-Gln (SEQ ID NO:86),Ala-Leu-Asp-Phe-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:87),Leu-Asp-Phe-Ser-Trp-Leu-Gln (SEQ ID NO:88),Leu-Asp-Phe-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:89),Thr-Ala-Leu-Asp-Tyr-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:90),Leu-Asp-Tyr-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:91),Thr-Ala-Leu-Asp-Tyr-Ser-Trp-Leu (SEQ ID NO:92),Ala-Leu-Asp-Tyr-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:93),Leu-Asp-Tyr-Ser-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:94),Leu-Asp-Tyr-Ser-Trp-Leu (SEQ ID NO:95),Thr-Ala-Leu-Asp-Tyr-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:96),Thr-Ala-Leu-Asp-Tyr-Ser-Trp-Leu-Gln (SEQ ID NO:97),Ala-Leu-Asp-Tyr-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:98),Leu-Asp-Tyr-Ser-Trp-Leu-Gln (SEQ ID NO:99),Leu-Asp-Tyr-Ser-Trp-Leu-Gln-Thr (SEQ ID NO:100),Thr-Ala-Leu-Asp-Trp-Ala-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:101),Leu-Asp-Trp-Ala-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:102),Thr-Ala-Leu-Asp-Trp-Ala-Trp-Leu (SEQ ID NO:103),Ala-Leu-Asp-Trp-Ala-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:104),Leu-Asp-Trp-Ala-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:105),Leu-Asp-Trp-Ala-Trp-Leu (SEQ ID NO:106),Thr-Ala-Leu-Asp-Trp-Ala-Trp-Leu-Gln-Thr (SEQ ID NO:107),Thr-Ala-Leu-Asp-Trp-Ala-Trp-Leu-Gln (SEQ ID NO:108),Ala-Leu-Asp-Trp-Ala-Trp-Leu-Gln-Thr (SEQ ID NO:109),Leu-Asp-Trp-Ala-Trp-Leu-Gln (SEQ ID NO:110),Leu-Asp-Trp-Ala-Trp-Leu-Gln-Thr (SEQ ID NO:111),Thr-Ala-Leu-Asp-Trp-Glu-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:112),Leu-Asp-Trp-Glu-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:113),Thr-Ala-Leu-Asp-Trp-Glu-Trp-Leu (SEQ ID NO:114),Ala-Leu-Asp-Trp-Glu-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:115),Leu-Asp-Trp-Glu-Trp-Leu-Gln-Thr-Glu (SEQ ID NO:116),Leu-Asp-Trp-Glu-Trp-Leu (SEQ ID NO:117),Thr-Ala-Leu-Asp-Trp-Glu-Trp-Leu-Gln-Thr (SEQ ID NO:118),Thr-Ala-Leu-Asp-Trp-Glu-Trp-Leu-Gln (SEQ ID NO:119),Ala-Leu-Asp-Trp-Glu-Trp-Leu-Gln-Thr (SEQ ID NO:120),Leu-Asp-Trp-Glu-Trp-Leu-Gln (SEQ ID NO:121), andLeu-Asp-Trp-Glu-Trp-Leu-Gln-Thr (SEQ ID NO:122).
 7. The method of claim1, wherein X_(a) consists of 6-12 amino acid residues.
 8. The method ofclaim 1, wherein X_(a) consists of 6-10 amino acid residues.
 9. Themethod of claim 1, wherein X_(a) comprises at least five basic aminoacid residues.
 10. The method of claim 1, wherein X_(a) comprises atleast five amino acid residues independently selected from L-arginine,D-arginine, L-lysine and D-lysine.
 11. The method of claim 1, whereinX_(a) is selected from the group consisting ofArg-Arg-Met-Lys-Trp-Lys-Lys (SEQ ID NO:123),Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO:124),D-Tyr-D-Gly-D-Arg-D-Lys-D-Lys-D-Arg-D-Arg-D-Gln-D-Arg-D-Arg-D-Arg (SEQID NO:125), Tyr-Ala-Arg-Lys-Ala-Arg-Arg-Gln-Ala-Arg-Arg (SEQ ID NO:126),D-Tyr-D-Ala-D-Arg-D-Lys-D-Ala-D-Arg-D-Arg-D-Gln-D-Ala-D-Arg-D-Arg (SEQID NO:127), Tyr-Ala-Arg-Ala-Ala-Arg-Arg-Ala-Ala-Arg-Arg (SEQ ID NO:128),D-Tyr-D-Ala-D-Arg-D-Ala-D-Ala-D-Arg-D-Arg-D-Ala-D-Ala-D-Arg-D-Arg (SEQID NO:129), D-Arg-D-Arg-D-Met-D-Lys-D-Trp-D-Lys-D-Lys (SEQ ID NO:130),Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO:149), Arg-Arg-Arg-Arg-Arg-Arg-Arg(SEQ ID NO:150), Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO:151),Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO:152),Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO:153),Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO:154),D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg (SEQ ID NO:155),D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg (SEQ ID NO:156),D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg (SEQ ID NO:157),D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg (SEQ ID NO:158),D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg (SEQ IDNO:159), andD-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg (SEQID NO:160).
 12. The method of claim 1, wherein the compound blocks theinteraction between one or more IKKs and NEMO.
 13. The method of claim12, wherein the IKK is selected from the group consisting of IKKα andIKKβ.
 14. The method of claim 1, wherein the compound modulatesNF-κB-dependent target gene expression.
 15. The method of claim 14,wherein the NF-κB-dependent target gene is E-selectin.
 16. The method ofclaim 1, wherein the compound modulates NF-κB induction.
 17. The methodof claim 1, wherein the compound inhibits osteoclast differentiation.18. The method of claim 1, wherein the cell is contacted in vivo. 19.The method of claim 1, wherein the cell is contacted in vitro.